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Ventricular vascular coupling in rodents and humans with spinal cord injury : a translational retrospective… Alanis, Guillermo A. 2019

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  VENTRICULAR VASCULAR COUPLING IN RODENTS AND HUMANS WITH SPINAL CORD INJURY: A TRANSLATIONAL RETROSPECTIVE STUDY by Guillermo A. Alanis B. Medicine., Universidad de Guadalajara, 2013  MASTER OF SCIENCE in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Kinesiology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2019 © Guillermo A. Alanis   ii    The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled:  Ventricular vascular coupling in rodents and humans with spinal cord injury: A translational retrospective study  submitted by Guillermo Adrian Alanis in partial fulfillment of the requirements for the degree of Master of Science in Kinesiology  Examining Committee: Christopher R West, Department of Cellular and Physiology Sciences, Southern Medical Program, UBC Supervisor  Aaron A Phillips, Department of Cellular and Physiology Sciences, University of Calgary Supervisory Committee Member  A William Sheel, School of Kinesiology, UBC Supervisory Committee Member         iii  Abstract  Spinal cord injury (SCI) is associated with cardiac atrophy, impaired systolic and diastolic function, and vascular stiffening. In able-bodied (AB) individuals, the heart and vasculature act in unison to ensure efficient coupling between the heart and the peripheral vasculature. The evaluation of the interaction between the vascular system and the heart is performed by measuring the vascular load imposed on the heart (arterial elastance), cardiac contractility (end-systolic elastance), and their ratio “ventricular-arterial coupling” (VAC). More specifically, arterial elastance (EA) is a parameter of the compliant properties of the arterial system and end-systolic elastance (Ees) determines the effectiveness of the heart as a pump. The VAC ratio is an important index of cardiac performance, linked to exercise capacity and predictor of both heart failure and cardiovascular (CV) mortality. Taken together these indices evaluate the mechanical efficiency of the cardiovascular system to meet the metabolic demands. A way to accurately evaluate this CV coupling is invasive and almost exclusively performed in animal models. However, a non-invasive approach to estimate VAC in the clinical scenario is through cardiac imaging and blood pressure measurement. In the field of SCI, research has only focused on evaluating these parameters in animal models, while in humans the heart and vasculature have been evaluated as independent units without investigating their “coupling.” The primary objective of this research was, therfore, to compare invasive and non-invasive parameters of cardiac systolic function in a validated SCI rodent model and translate these findings to humans. Additionally, to determine the parameter that better reflects the impaired systolic function in SCI, I will investigate two non-invasive approaches to assess systolic function and their iv  vascular “coupling” in elite athletes with chronic cervical SCI, non-athlete chronic cervical SCI individuals and AB.                 v  Lay summary  After spinal cord injury, there are changes in the heart and blood vessels that affect their normal function. In this study, we looked at these changes, how the heart and blood vessels interact, and the effect of prolonged exercise on this interaction. We found that individuals with SCI had a lower estimated cardiac function and higher resistance to flow in their blood vessels which negatively impacts the ejection of blood out of the heart. We also found that regular exercise may improve  cardiac pumping capacity due to better blood vessel function.  The primary parameter used to evaluate cardiac function in humans was further validated in rats with SCI, where it was highly correlated with invasive parameters of cardiac contractility.           vi  Preface  Chapter 2 of this thesis was approved by the University of British Columbia clinical research ethics board (H17-00463). The retrospective animal data analysis was obtained from a study approved by the UBC Animal Care Committee (A18-0344). The human ultrasound imaging in Chapter 2 was conducted by Dr. A Williams and C. Gee organized and supported the athlete group testing. I performed all image analysis and recruitment of the able-bodied and non-athletic SCI cohort. The animal data from which I performed a retrospective validation was derived from a study conducted by B. Hayes. I analyzed the ultrasound and pressure-volume data. The formulas to calculate my main outcome variables were developed by C. Chen-Huan and A. Young and cited where appropriate. A part of the human findings was presented in an oral presentation at a national conference. None of this work has been published in peer-reviewed journals.            vii  Table of contents  Abstract ................................................................................................................................. iii Lay summary .......................................................................................................................... v Preface....................................................................................................................................vi Table of contents .................................................................................................................. vii List of tables ...........................................................................................................................ix List of figures .......................................................................................................................... x List of abbreviations ............................................................................................................ xii Acknowledgments................................................................................................................xiv Dedication ............................................................................................................................. xv Chapter 1.  Literature review .................................................................................................. 1 1.1 The heart .................................................................................................................... 1 1.2 The circulatory system............................................................................................... 6 1.3 Blood pressure ......................................................................................................... 11 1.4 The autonomic nervous system ............................................................................... 16 1.5 The cardiac cycle ..................................................................................................... 20 1.6 The pressure-volume relationship ........................................................................... 23 1.7 Preload ..................................................................................................................... 25 1.8 Afterload .................................................................................................................. 26 1.9 End-systolic pressure-volume relationship .............................................................. 27 1.10 End-systolic elastance............................................................................................ 28 1.11 Ventricular stiffness index ..................................................................................... 30 1.12 Arterial elastance ................................................................................................... 31 1.13 Ventricular-arterial coupling ................................................................................. 33 1.14 Cardiovascular pathophysiology in SCI ................................................................ 35 1.15 Aims....................................................................................................................... 46 1.16 Hypotheses............................................................................................................. 46 Chapter 2: Non-invasive ventricular-arterial couplings in humans and animals with SCI... 47 2.1 Brief introduction .................................................................................................... 47 2.2 Methods ................................................................................................................... 49 2.3 Results ..................................................................................................................... 58 viii  2.4 Discussion ................................................................................................................ 69 Chapter 3. Conclusion ........................................................................................................... 83 3.1 Implications and future directions ........................................................................... 83 3.2 Study strengths and limitations................................................................................ 83 Bibliography ......................................................................................................................... 85                         ix  List of tables  Table 2.1 Demographic and physical activity characteristics ........................................... 59 Table 2.2 Hemodynamic and vascular structure and function between groups. ............... 60 Table 2.3 Left ventricular structure and volume between able-bodied, athletes and non-athletes with SCI. ............................................................................................................... 62 Table 2.4 Non-invasive ventricular-arterial coupling calculated by Ees(sb) and VSI. ..... 64 Table 2.5 Catheter derived parameter of systolic function and ventricular-arterial coupling between sham and SCI rats. ............................................................................................... 65 Table 2.6 Baseline and termination pulmonary artery Doppler parameters. .................... 67               x  List of figures  Figure 1.1  Interaction between the cardiac and the venous function curve to determine cardiac output. Change in cardiac output (closed blue circle) from baseline (closed red circle, A) after increasing contractility (dashed line). Increase in cardiac output with an increase in venous return (open red circle, B)........................................................................................... 14 Figure 1.2 Wiggers diagram illustrating the correlation between the mechanical and electrical events during the cardiac cycle of the left ventricle. ............................................... 22 Figure 1.3 a) Pressure-volume loop, b) Time-pressure graph representing changes in pressure and volume during the cardiac cycle. ....................................................................... 23 Figure 1.4 Stroke volume (SV) measurement in a pressure-volume loop by the difference between end-diastolic volume (EDV) and end-systolic volume (ESV). ................................ 25 Figure 1.5 End-systolic elastance (Ees) obtained from a series of pressure-volume loops under different preload conditions. ......................................................................................... 28 Figure 1.6 The intercept of end-systolic elastance (Ees) and arterial elastance (EA) representing ventricular-arterial coupling (VAC). .................................................................. 32 Figure 1.7 Stroke work (SW) and the potential energy (PE) in a pressure-volume loop. ..... 34 Figure 1.8 a) Increased vasoconstrictor response of femoral artery rings in SCI rats (open dots) after increasing phenylephrine infusion compared to controls (closed dots) Source: Alan et al.141. b) Enhanced vasoconstrictor response after phenylephrine infusion (PE) in rats that underwent repetitive colorectal distension (SCI-CRD) to induce autonomic dysreflexia, compared to SCI controls (SCI). Source: Lee et a.140. ............................................................ 41 xi  Figure 1.9 Relationship between HR and oxygen consumption (VO2) in able-bodied (AB), low thoracic paraplegic (MLPara), high thoracic paraplegic (HLPara) and tetraplegic (Tetra). Source: Schmid et al. 199895. .................................................................................................. 43 Figure 2.1 Peak blood velocity (PBV), time-to-peak (TTP) and ventricular stiffness index (VSI) at baseline and termination in sham (blue) and spinal cord injury (SCI) (green) animals. *Significant post-hoc comparison at that time point following an interaction effect (p<0.05); † significant main effect for time (p<0.05). ............................................................ 66 Figure 2.2 Correlation between maximal rate pressure change-EDV (dP/dt max-EDV) and preload recruitable stroke work (PRSW) and ventricular stiffness index (VSI). Closed blue and green circles represent sham and SCI animals, respectively. Significant relationship p<0.05. .................................................................................................................................... 68           xii  List of abbreviations  2D – two-dimensional Ach – acetylcholine  AD – autonomic dysreflexia  ANS – autonomic nervous system AP – action potential  AV – atrioventricular BMI – body mass index  BP – blood pressure  BSA – body surface area CFA – common femoral artery  cGMP – cyclic guanosine monophosphate  CV – cardiovascular  CVLM – caudal ventrolateral medulla  CVP – central venous pressure  DBP – diastolic blood pressure  dP/dtmax – maximal rising rate of LV pressure during isovolumetric contraction  dQ/dt max – maximal rate change of blood flow  E – mitral early filling peak velocity Ea(Simp) – arterial elastance derived from Simpson´s biplane ECM – extracellular matrix EDV end-diastolic volume  EF – ejection fraction  Ees – end-systolic elastance  Ees(sb) – single-beat Ees Em – annular early diastolic tissue velocity  ESBP – end-systolic blood pressure ESPVR – end-systolic pressure-volume relationship  ESV – end-systolic volume  FMD – flow-mediated dilation  HFpEF – heart failure with preserved ejection fraction LVIDd – internal diameter at end-diastole MAP – mean arterial pressure  MMP9 – matrix metalloproteinase 9  NE – norepinephrine  NO – nitric oxide  NTS – nucleus of the tractus solitarius  OH – orthostatic hypotension  PBV – peak blood velocity  xiii  PE – potential energy  PEP – pre-ejection period PNS – parasympathetic nervous system  PP – pulse pressure PRSW – preload recruitable stroke work  PV loops – pressure-volume loops PVA – pressure-volume area  Qmax – maximal blood flow in the descending aorta  RA – right atrium RAS – renin-angiotensin system  RV – right ventricle RVLM – rostral ventrolateral medulla  SA – sinoatrial SBP – systolic blood pressure  SCI-CRD – colorectal distention  SFA – superficial femoral artery  SMC – smooth muscle cells  SV – stroke volume  SW – the stroke work TIMP1 – tissue inhibitor of metalloproteinase 1  TPR – total peripheral resistance  TTP – time to peak velocity  VAC – ventricular-arterial coupling  VAC(VSI) – ventricular arterial coupling derived from ventricular stiffness index VSI – ventricular stiffness index            xiv  Acknowledgments  I would like to show my appreciation and gratitude to my supervisor Dr. Christopher West for his guidance and support throughout these years. I would also like to thank my former laboratory colleagues in Mexico who encouraged and supported me in my Masters' application.  I would like to thank the members of the West lab, Dr. Alex M. Williams, Cameron Gee, Mary Fossey, Erin Erskine, and Brian Hayes for their constant support during my project. I am grateful to the members of my committee, Dr. Aaron Phillips and Dr. William Sheel, for their valuable advice and contributions to this project. I would like to extend my acknowledgements to my professors, staff, and colleagues from University of British Columbia; the David W. Strangway Fellowship, and the Shaughnessy Hospital Volunteer Society for their financial support.  Finally, I want to acknowledge the people in this study for their participation and interest in contributing to science.          xv  Dedication  To my family.                           1  Chapter 1.  Literature review  1.1 The heart  1.1.1 Anatomy of the heart  The heart is a cone-shaped organ orientated in an oblique position (two thirds into the left midline) in the center of the thoracic cavity, specifically the mediastinum, posterior to the sternum and above the superior surface of the diaphragm. The base of the heart is located below the third rib, and the apex projects anteriorly to the left mid-clavicular line. The exterior surface of the heart is covered by a serous membrane of dense connective tissue called the pericardium, that at the same time, is composed by two different but continuous layers called the parietal (outer) and visceral (internal) pericardium. The facing surface of these layers forms the pericardial space which is filled with a lubricant fluid secreted by the mesothelial cells that reduce friction between these two layers during cyclic contractions of the heart1. The size of the heart depends on many factors, but it weighs approximately 0.4% to 0.5% of total body mass. The inner structure of the heart is composed of four closed cavities or chambers. Which main function is to collect blood coming from peripheral tissue and pump it to the lungs to be oxygenated. The heart then collects the oxygenated blood from the pulmonary veins to be pumped out to the rest of the body. The chamber walls consist of striated and involuntary cardiac muscle, histologically divided into three main layers, from external to internal, called the epicardium, the myocardium, and the endocardium. The epicardium, also known as the visceral pericardium, is a serous membrane that envelops the outer surface of the heart and is composed by connective and adipose tissue, and the 2  mesothelium2. The myocardium, found in both the atria and the ventricle, is the contractile layer of the heart and is the thickest layer given its pressure-generating capacity. This contractile layer consists of elongated and branched cardiac muscle cells (cardiomyocytes) bound by intercalated discs that propagate action potentials and allows them to contract uniformly. The average diameter of the cardiomyocytes ranges from 10-35 µm, and the length ranges from 85 to 120 µm3, 4. Unlike the skeletal muscle, the nucleus of the cardiomyocyte lies within the center of the cell. Some might be binucleated, and the perinuclear region is free of myofibrils which exhibit high concentrations of surrounding mitochondria (up to 40% of the cell´s volume)3. The most inner layer of the heart, the endocardium, is composed by sublayers named the endothelial, subendothelial and subendocardial layers. The endothelial layer, or endothelium, is formed by a single layer of epithelial cells that are an extension of the great vessels and cover the inner structures of the heart, such as the atrioventricular and semilunar valves, the inner surface of the chambers, the chordae tendinae, and the papillary muscles5. Beneath the endothelium is the subendothelial layer which is composed of connective tissue containing fibroblasts, elastic and collagen fibers, and the subendocardial layer which consists of deep connective tissue that merges with the myocardium and allows for the optimal transmission of the electrical activity2. In terms of the cellular makeup of the adult mammalian heart, it has been stated that the cardiomyocytes constitute the majority of the cardiac mass, but they account for ~30% of the cells of the heart6. Of the remaining cell types, the endothelial cells (EC) are the most common cell types (~51%) with ratios reported between EC and cardiomyocytes of 3:1. Following in frequency, are the cardiac fibroblasts (~15%), leucocytes (7-10%), and 3  pericytes (~5%)7. These different cell types in the human heart are surrounded and supported by different functional and structural proteins that form the extracellular matrix (ECM). The ECM consists of acellular components such as collagen, proteoglycans, glycoproteins, proteases, cytokines, and matrikines. Together, these components of the ECM serve essential functions including the distribution of mechanical forces, response to harmful stimuli, growth, degradation, and signaling8. The heart consists of two upper chambers, the right and left atria, which are separated by the interatrial septum and collect blood from the venous systemic and pulmonary circulation; while lower chambers, the right and left ventricles are separated by the interventricular septum and pump out blood received from their corresponding atria. A structure situated in the base of the heart, the coronary sulcus, separates the atria from the ventricles and in the posterior face of the heart, contains the coronary sinus that collects the venous blood of the myocardium and drains it into the right atrium (RA). Inside the heart, the atria and the ventricle are separated by the atrioventricular valves which prevent backflow from the ventricle to their corresponding atria, assuring a proper unidirectional blood flow9.   Right atrium and ventricle The RA is located in the upper right part of the heart, above the right ventricle (RV), and receives deoxygenated blood from the venous system through the superior and inferior vena cava, and by the myocardium itself via the coronary sinus. The RA is divided into three anatomical regions named the posterior, anterior, and interatrial septum. The posterior region of the RA, which is characterized by a smooth muscle wall, contains the fossa ovalis (embryologic remnant) and two key structures of the electrical conduction system of the heart, the sinoatrial (SA) and atrioventricular (AV) node. The SA node is located within the 4  atrial wall between the myocardium and the epicardium in the superior portion of the RA and next to the lateral border of the superior vena1. This node is considered the pacemaker of the heart due to its capability of generating spontaneous action potentials that are propagated by the conducting system to create rhythmic contractions in a range of 60-100 bpm10. However, specialized conduction cells found in the AV node, the bundle of His and its branches can produce spontaneous depolarizing action potentials but at a lower range within 25-55 bpm. Called ventricular escape rhythm that can maintain some degree of cardiac output in the case of an inappropriate function of the SA or AV nodes. Furthermore, the myocardium itself can be an unusual source of spontaneous depolarization (ectopic pacemaker) that can produce extra beats or set a new contractility rhythm, also within 25-55 bpm9.  The anterior inner portion of the RA is separated from the posterior portion by a muscle band named crista terminalis, from which horizontal muscle fibers arise arranged in comb fashion called pectinate muscles and give origin to the right atrial appendage or auricle. The interatrial septum region contains a remnant of the fetal circulation, the fossa ovalis, which in the adult heart appears as a central depression11. The RA is separated from the RV by the tricuspid valve. As the name suggests, it is formed by three leaflets called anterior (superior), posterior (inferior) and septal. The anterior is the largest leaflet and is attached to the medial septum and the anterior free wall. The posterior is attached to the lateral free wall and the posterior part of the septum, while the septal leaflet is attached to the medial part of the septum and the annulus of the orifice9. The base of these leaflets is anchored to a rigid and fibrous structure called the cardiac skeleton 12. Each of these leaflets is fixed to the ventricular wall by the papillary muscles (anterior, posterior and septal papillary muscles) 5  through fibrous cords called chordae tendineae. Although, they can also be attached directly to the myocardial wall, especially in the anterior and septal regions of the ventricle9. The RV, unlike the LV, is a crescent-shaped structure that is located inferiorly and anteriorly with respect to the RA, and to the right of the LV13. Functionally, the RV can be divided into the inflow and outflow tract. The inflow tract is where the blood flow enters from the atria. It is formed by the annulus and leaflets of the tricuspid valve, papillary muscles, and the rough trabeculated apex (intraluminal projections of the myocardium). On the other hand, the outflow tract is characterized by smooth surface due to the absence of trabeculation and formed by the infundibulum (the area from which the pulmonary artery arises) and the septomarginal trabecula (moderator band)9. Left atrium and ventricle  The LA is located in the midline of the thoracic cavity, superior to the LV and behind the RA. Similarly, to the RA, the LA presents an auricular appendage but with fewer pectinate muscles, a greater smooth internal surface and a thicker wall (due to higher pressures compared to the RA). The LA receives oxygenated blood from the lungs by two pairs of pulmonary veins, the right and left pulmonary veins, inserted into the posterior and lateral region of the atria. The LA is separated from the LV by a 2-cuspid valve called mitral or bicuspid valve which is attached to the anterior and posterior papillary muscles1.  In contrast to the RV, the myocardium of the LV can be 3 times thicker, due to higher pressures in the systemic circulation (120 mmHg) compared to the pulmonary circulation (25 mmHg), and has a thinner trabeculation pattern1. This trabeculation pattern, which is a remnant of the embryogenic development of the heart14, helps the ventricular filling by 6  increasing its compliance. A greater trabeculation pattern makes the RV more compliant and suitable for low filling pressures15. 1.2 The circulatory system  1.2.1 Structure of the circulatory system  The circulatory system is a closed vascular circuit formed by tubes of connective tissue and smooth muscle fibers. The primary goal of the circulation is to supply the body with oxygen (O2), transport hormones and nutrients, and removal of metabolic by-products. This tubing system can be divided into the venous and arterial system, consisting of arteries and veins of various diameters, layers, and pressure-flow characteristics. The circulation within the arterial system can be classified into the pulmonary and the systemic (peripheral) circulation. In the peripheral circulation, the different types of arteries such as elastic (conducting), muscular (distributing), arterioles and capillaries serve various functions and exhibit different structures and diameters. The arterial system is characterized for being a low volume (10-15% of the total blood volume) and high-pressure system, unlike the venous system which handles up to 60% of the total blood volume at low pressures16. Arteries and veins are structured in three different layers, except for the capillaries and venules which only present one layer (endothelium), identified as the tunica intima, tunica media, and tunica adventitia. The tunica intima is the most internal layer and is formed by a single layer of endothelial cells that cover the lumen of the vessel called endothelium, a subendothelial layer of loose connective tissue, and an elastin-based fenestrated band circumferentially surrounding the lumen called the internal elastic lamina. The endothelium acts as a physical barrier from other blood compounds, produces vasoactive substances that 7  prevent coagulation, regulate vascular tone, transport of specific molecules, and proteins across the vascular wall and mediate inflammatory response via leukocyte recruitment17. The middle layer is called the tunica media, and it is composed of smooth muscle cells (SMC) and ECM containing elastin, which confers elastic properties to buffer blood flow during contraction and recoil during diastole, and collagen that confers structural strength and support to the vascular wall. Elastin is the major protein of the arterial wall of elastic arteries making up 50% of the dry mass in the case of the aorta18. This protein is circumferentially organized around the blood vessel forming a structure called elastic lamellae that alternates with SMC and form the lamellar units. Depending on the type of artery or vein, the proportion of lamellar units will differ as well as the amount of SMC19. The outermost layer is the tunica adventitia and gives the vessel wall structural support due to the composition of its connective tissue mainly consisting of type 1 collagen, elastic fibers, and fibroblasts. In the case of large arteries, the cells of the adventitia layer cannot be nourished and oxygenated by the luminal O2. Thus a network of capillaries called the vasa vasorum, perfuse the cells within tunica adventitia in large blood vessels. Compared to the arteries, the vasa vasorum is more developed in veins as a compensation for the lower levels of oxygen in mixed venous blood (15 mL O2  per 100 mL blood) compared to arterial blood (20 mL O2  per 100 mL blood) at rest4. In the arterial system, arteries are broadly classified into elastic and muscular arteries. The elastic arteries are the greatest in diameter (>10 mm) and are the closest to the heart (aorta, carotid, pulmonary arteries). These arteries have an abundant proportion of elastic components that confers cushioning and recoil properties to produce constant blood flow. The muscular arteries are considered medium-sized arteries (1-10 mm) and they have a 8  higher proportion of SMC compared to elastic arteries which give them a high capacity to change their internal diameter in response to sympathetic stimulation. Examples of muscular arteries include the femoral, brachial and radial arteries.  Following in diameter, are the small arteries (0.1-1 mm) that supply blood to arterioles and are also capable of controlling blood flow by vasodilation or vasoconstriction. The arterioles (100 µm-10 µm) are the last branch of arteries and mark the beginning of the microvasculature. They are composed of one or two layers SMC, they lack elastic components, and represent the primary determinant of blood flow resistance and therefore, blood pressure. The arterioles further branch into smaller vessels called metarterioles which are controlled by encircling bands of SMC named precapillary sphincters. These respond to local stimulus, vasoactive substances, and innervation from the SNS20, 21. Finally, the metarterioles give origin to the smallest blood vessels, the capillaries22. A single layer of EC composes the capillaries; they are 5 µm in diameter at the arterial end and 9 µm at the venous end. These dimensions and the 1 µm thickness of the capillary wall, permit the passage of red blood cells in a single line and allow for O2/CO2 exchange, as well as nutrients/waste products by diffusion or endocytosis, depending on the size of the molecule23. The EC of the capillaries and venules are wrapped by mesenchymal cells called pericytes that by contraction of their cytoskeleton or by secretion of vasoactive agents regulate blood flow in the vasculature, help maintain the blood-brain barrier integrity in the CNS24 and assist in the regeneration process in other tissues3. At the end of the capillaries, the beginning of the venous system starts with the venules. The venules are slightly thicker than capillaries and have a small amount of SMC in the media layer, but they are still sensitive to adrenergic action that produces venoconstriction23. Following the 9  venules, the diameter and thickness of the venous wall will continue to increase until reaching an approximate diameter of 3 cm and 1.5 mm thickness in the vena cava that ultimately ends in the RA. 1.2.2 Control of vascular tone  The primary determinant of blood pressure is the resistance to flow presented by the state of constriction or “tone” of the SMC that regulate the arteriolar diameter. The tone of the arterioles is governed by factors that can be categorized into myogenic, neural, and hormonal factors25.  The myogenic mechanisms act independently of sympathetic stimulation or hormones and is influenced by the metabolic state of the specific organ, the automatic myogenic response, the influence from the EC, and the action of vasodilator substances (nitric oxide, prostaglandins, histamine, bradykinin, endothelium-derived hyperpolarizing factor) and vasoconstrictor substances (thromboxane, endothelin). The arterioles also exhibit an active and passive response to changes exerted on their vascular wall (transmural pressure). For instance, an initial increase in blood flow will distend the vascular wall with a rise in transmural pressure which will trigger an active vasoconstrictor response to reverse the pressure increase. Similarly, a decrease in transmural pressure will passively reduce the diameter and increase the tone to maintain the previous baseline pressure. These automatic adjustments in pressure, diameter and tone are part of the myogenic response25. The endothelium also plays a crucial role in regulating the blood vessel’s wall tone through the release of vasodilators including vasoactive intestinal peptide and nitric oxide (NO) stimulated by acetylcholine or shear stress (frictional force against the endothelium). 10  NO is a lipid-soluble peptide produced from the substrate L-arginine by NO synthase that once formed, diffuses through the subendothelial layer and internal elastic lamina to reach the SMC. Once coupled to the receptors on the SMC, NO increases the production of cyclic guanosine monophosphate (cGMP) which causes a decrease in intracellular calcium concentration causing relaxation of the SMC and vasodilation26.   The peripheral blood vessels are almost exclusively innervated by the SNS, except for the arteries in the erectile tissue27, which increases the tone of the blood vessels by activating its adrenergic receptors in the SMC within the tunica media, hence the greater SMC present within a blood vessel, the more responsive it is to sympathetic stimulus. The contraction of the SMC occurs by the action of the second messenger inositol triphosphate (IP3) that increases intracellular calcium and activates the coupling sites of the thin and thick filaments of the SMC fibers. The neural control plays a major role in regulating arterial tone by either increasing the activity of the SNS firing rate, with subsequent vasoconstriction or by a reduction of the firing rate which produces vasodilation25. The hormonal control of the vascular tone is mainly regulated by the action the catecholamines (epinephrine and norepinephrine) released by the adrenal medulla in cases of intense exercise or hemorrhagic shock. The catecholamines released from the adrenal glands are secreted by stimulation of the SNS. These activate vascular adrenergic receptors (α-receptors) and cardiac receptors (β1 receptors) producing vasoconstriction and an increase in contractility and HR, respectively. However, due to their short two-minute half-life, these effects are transient and brief. Other hormones, like vasopressin and angiotensin II, also have vasoactive properties but are more important in the long-term regulation of blood pressure. Vasopressin, also known as the anti-diuretic hormone, is released from the posterior pituitary 11  gland in cases of blood volume loss or increased extracellular osmolality. It has a potent vasoconstrictor effect on the arterioles; however, it is not considered to have an active role in regulating vascular tone in normal conditions. Similarly, angiotensin II is a hormone that regulates the sodium balance in body fluids by acting on kidney function and even though angiotensin II is also a potent vasoconstrictor like vasopressin, it is not thought to be involved in the long-term regulation of vascular tone28. 1.3 Blood pressure  1.3.1 Overview  The blood pressure (BP) a frequently measured clinical variable that gives us some basic information about the overall status of the cardiovascular (CV) system. BP is defined as the force applied to the arterial wall by the blood flow ejected during the cyclical contractions of the LV, causing oscillations in the pressure level. The highest peak of these oscillations is called systolic blood pressure (SBP), the lowest level is called diastolic blood pressure (DBP) and the time-averaged pressure in one cardiac cycle, is called mean arterial pressure (MAP)29.  1.3.2 Mean arterial pressure  The MAP is the effective arterial pressure that perfuses the organs and can be defined as the product of CO and total peripheral resistance (TPR). In the clinical scenario, it is mathematically estimated with the formula MAP= DBP + 1/3 of pulse pressure (the difference between SBP and DBP). The MAP is closely related to CO and the TPR in the vascular system. For example, an increase CO with a constant TPR will increase MAP, while a decrease in TPR will negatively affect MAP. Since the main location of vascular resistance 12  is found in the arterioles, TPR will be influenced by constriction or dilation of the arterioles by the SNS and the age-related loss of endothelium-dependent vasodilation30. On the other hand, increases or decreases in heart rate (HR) and stroke volume (SV) will determine CO and subsequently MAP31.  1.3.3 Cardiac output  Cardiac output is an important measure of cardiac function because it represents the total amount of blood available to satisfy the body’s metabolic demands. It is the blood volume ejected by the LV in one minute and is determined by SV and HR. For this reason, the capability to increase several times from rest during exercise is essential for exercise performance. In untrained young individuals, the cardiac output can increase up to 5 times to satisfy metabolic demands, from 5 L/min to 25 L/min by parallel increases in HR and SV32. The inherent capability of the CO to increase and the importance of venous return can be explained by the Frank-Starling mechanism. The essence of this mechanism was first described by Carl Ludwig in 1856 with an isolated frog heart. He stated that “. . . A strong heart that is filled with blood empties itself more or less completely, in other words, [filling of the heart with blood] changes the extent of contractile power”33. This observation laid the foundations of the future research conducted by Otto Frank in 1895, also with frog´s hearts, in which he observed that increasing the filling of the heart would increase the diastolic pressure with greater maximal isovolumetric pressure or force of contraction34. Subsequently, in 1914, Ernst Starling and colleagues35 experimented with isolated canine hearts preparations that allowed them to manipulate inflow to the heart (preload) and arterial resistance (afterload). They observed that by increasing the ventricle´s filling pressure and at 13  a constant afterload, the SV increased; conversely when increasing afterload, the heart would not empty itself as effectively with greater remaining volume at end-systole. Nevertheless, the accumulated end-diastolic volume in the following cardiac cycle increased the ventricle’s filling pressure, subsequently increasing the force of contraction and restoring the original SV. In summary, these experiments demonstrated that the ventricular force of contraction increases proportionally with a greater stretching of the myocardial fibers before contraction due to greater ventricular filling. This gave origin to the Frank-Starling´s mechanism or “law of the heart”36. The Frank-Starling mechanism is based on the length-tension relationship that relies on the length of the contractile units of the heart, the sarcomeres, which will determine the alignment and coupling of the thin (actin) and thick (myosin) contractile filaments. The length of the sarcomere is measured by the distance between its Z-lines, which changes during systole and diastole and ranges from 1.6 to 2.2 µm of length37. The optimal alignment of actin and myosin to generate a contraction is found at a sarcomere length between 2.2 and 2.3 µm38. A length greater than this “optimal” point would mean that only fewer myosin heads would be aligned with their corresponding actin filaments, thus generating a weaker contraction. Similarly, a reduced sarcomere length creates an overlapping of these filaments also creating a weaker contraction.  The degree of venous return is determined by the degree of resistance to flow back to the heart and the mean circulatory filling pressure (MCFP)39 . The resistance of venous return is determined by the pressure difference between the RA and the venous compartment, in other words, a greater difference between the RA and the venous circulation will reduce the resistance of blood flow back to the heart increasing ventricular filling. This pressure 14  difference between the RA and the venous compartment can be achieved by increasing pressure in the venous circulation by either increasing volume (e.g., blood redistribution) or decreasing the vascular wall compliance (venoconstriction). However, CO is also affected by afterload and by cardiac function39 through changes in contractility and ventricular compliance. Nevertheless, if we were to plot a cardiac function curve and a venous function curve together (Figure 1.1), we would observe that by solely increasing the contractility of the heart at a constant central venous pressure (CVP) (dashed blue line) we would not have a significant increase CO (closed blue circle). Conversely, if there is an increase in venous return (greater CVP), we would observe a greater increase in CO (point B, open red circle). This response illustrates the importance of the Frank-Starling mechanism in determining CO39.   Figure 1.1  Interaction between the cardiac and the venous function curve to determine cardiac output. Change in cardiac output (closed blue circle) from baseline (closed red circle, A) after increasing contractility (dashed line). Increase in cardiac output with an increase in venous return (open red circle, B). 15  1.3.4 Peripheral vascular resistance  The objective of the arterial circulatory system is to conduct blood from the heart to the tissue at a constant flow through a tubing system with different diameters and pressures. As the diameter of the arteries progressively decreases, the resistance to flow increases. As a result, BP drops proportionally to the increased resistance within each vascular segment. For example, at the level of the aorta the mean pressure is ~100 mmHg, but as the blood flow advances through the small arteries, arterioles, and capillaries the pressure drops continuously down to an average of 25 mmHg at the capillary level31, a pressure low enough to allow the transport of molecules between the tissue and the circulation40. The pressure continues to decrease as the blood flows back to the heart where the pressure at the RA is nearly zero31. Physically, this gradual pressure decrease can be explained by hydraulic physical laws, such as the Law of Poiseuille. Poiseuille´s law describes the relationship of the three factors determining the resistance to flow and includes the fluid viscosity, the length and the radius of the vessel with the following formula41: Resistance = 8 * viscosity * length / π*radius4 This law states that the resistance to flow is proportional to blood viscosity, vessel length, and radius. However, the resistance to flow is affected by the vessel radius to the fourth power. For example, if the radius decreases by half, the resistance will increase 16 times and since the arterioles are the main location of blood flow resistance, MAP and therefore, organ perfusion will depend on the vasodilation or vasoconstriction at the arteriolar level. 16  1.4 The autonomic nervous system  1.4.1 Overview  The ANS is a specialized part of the nervous system that controls several of the most basic internal and unconscious visceral functions of our body, such as gastrointestinal motility, urinary tract function, temperature regulation, cardiorespiratory response to exercise, etc. These functions are regulated by visceral reflexes coming from the target organ, synapse with neurons in the dorsal root ganglion which relay sensory information via the spinal cord to the brainstem or the hypothalamus, and back to the organ. The body’s nervous system can be divided into enteric (neural control of digestive organs), and the ANS further divided into sympathetic and parasympathetic nervous systems. The enteric nervous system is a complex network of neurons, interneurons and motor neurons around the gastrointestinal tract that has a certain level of independence, but it is mostly controlled by the sympathetic and parasympathetic systems 42. The sympathetic and parasympathetic constitute the automatized neural control system that is responsible for creating physiological changes for a “fight” or “flight” response that innervates organs other than the skeletal muscle40, 43, its functions will be discussed in more detail since they are essential for the regulation of cardiovascular homeostasis. 1.4.2 Anatomy of the sympathetic nervous system  The excitatory function of the ANS is carried out by the SNS. The origin of the excitatory signal is originated near the pial surface of the medulla in the rostral ventrolateral medulla (RVLM). The RVLM can receive direct stimulation (e.g. CO2, hypoxia), excitatory (e.g. brainstem, cerebral cortex, etc.) or inhibitory input (e.g. hypothalamus, cardiopulmonary 17  baroreceptors). The neurons of the RVLM are glutamatergic (excitatory) neurons that can stimulate sympathetic preganglionic neurons (SPN) ̵ located in the intermediolateral horn of the spinal cord ̵ by increasing tonic excitation to the heart and peripheral vasculature or can inhibit them by decreasing their firing rate by inhibitory neurons of the CVLM. The input from the RVLM travel dorsally down the spinal cord, descend to the intermediolateral horn of the gray matter in the spinal cord where they synapse to SPNs. Subsequently, SPNs synapse with postganglionic neurons whose axons exit by the ventral roots of the spinal cord where they can synapse directly to sympathetic ganglia, enter the closest paravertebral ganglion where they synapse with postganglionic neurons (between the T1 and L2 spinal level)or they can synapse directly with the target organ (specifically in the case of the adrenal gland)40,43.  1.4.3 Anatomy of the parasympathetic nervous system  The organization of the parasympathetic nervous system (PNS) is different from the SNS. The location of the preganglionic cell bodies of the PNS is in the cranial nerves and the sacral spinal cord regions (S2-S4). Unlike the SNS, the axons of the preganglionic neurons will synapse the postganglionic neurons in terminal ganglia within the wall of the organ. The output from the PNS is performed via the oculomotor cranial nerve (CN III), facial nerve (CN VII), the glossopharyngeal nerve (CN IX), the vagus nerve (CN X) and neurons originated from the second to fourth sacral vertebrae. Of these, the vagus nerve is the primary output of this system innervating the organs in the thorax, abdomen and reproductive system40.   18  1.4.4 Function of the sympathetic and parasympathetic nervous system  As previously stated, the SNS is in charge of the “fight” or “flight” response. In situations of mental or physical “stress,” the SNS induces functional changes such as increasing HR, BP, and heart contractility, releasing of energy substrates to the bloodstream, and increasing skeletal muscle blood flow to respond adequately to the stressful situation40. In normal daily life situations, the internal organs are in a permanent balance between inhibition and stimulation, by both the SNS and PNS via two main transmitters, ACh and norepinephrine (NE). The nerve fibers that secrete NE are called adrenergic, and those that secrete ACh are called cholinergic. All the preganglionic fibers from both the SNS and PNS and all of the postganglionic parasympathetic nerve fibers are cholinergic, while most of the postganglionic SNS fibers are adrenergic, except for the sympathetic cholinergic fibers that innervate the sweat glands and the piloerector muscles44.  Whether there is a stimulatory or inhibitory response, it will depend on the cellular receptor activated. The cholinergic receptors include the muscarinic and nicotinic receptors, both activated by ACh, and the adrenergic receptors include the α (α1 and α2) and β (β1, β2, and β3) receptors that are activated by both norepinephrine anα1d epinephrine. The concentration and the affinity of these receptors will determine the degree of response of the target organ. For example, the α1 receptors on the SMC of the vascular wall have more affinity to NE, compared to β1 receptors in the heart. While β1 receptors are more sensitive to epinephrine and less sensitive to norepinephrine40.   19  1.4.5 Autonomic control of the heart  Intrinsic and extrinsic mechanisms control the function of the heart. The intrinsic regulation of the heart is usually performed by the SA node, which given its self-firing properties gives the heart an automated and independent mechanism to maintain a stable HR at approximately 70 bpm at rest under the influence of parasympathetic tone. Extrinsically, the function of the heart is also regulated by several parts of the insular cortex (limbic system), the forebrain and mainly the brainstem via the autonomic nervous system (ANS) through the sympathetic (excitatory) or the parasympathetic (inhibitory) system32. The brainstem receives regulatory afferent signals from the peripheral vasculature (arterial baroreceptors and chemoreceptors) previously integrated into the nucleus of the tractus solitarius (NTS) to maintain hemodynamic homeostasis and respond to metabolic demands. In the case of a direct cardio accelerator stimulus, there is an increased stimulation by the SNS. The axons of the RVLM travel dorsally down the spinal cord, descend to the intermediolateral horn of the gray matter in the spinal cord where they synapse onto  the preganglionic neurons, then the axons of the preganglionic neurons exit by the ventral roots of the spinal cord and synapse with the postganglionic neurons in the ganglia located between the 1st to 5th thoracic segments (T1-T5). The axons of the sympathetic postganglionic neurons innervate the SA and AV node, the atrial muscle and the ventricles, and release norepinephrine which activate adrenergic receptors increasing the frequency of contraction (chronotropism), transmission of the conductive system (dromotropy), myocardial relaxation during diastole (lusitropism), and contractility (inotropism)9.  Sensory input from peripheral receptors (afferent stimuli) can also regulate the activity of the heart and blood vessels. For instance, the baroreceptors located broadly in the 20  vascular wall, but in a major concentration in the carotid sinus and the aortic arch, detect changes in blood pressure. These baroreceptors are mechanoreceptors that detect stretching of the vascular wall, which in the case of increased pressure will cause an increase in their signal firing rate conducted by the glossopharyngeal nerve (in the case of carotid baroreceptors) and the vagus nerve (in the case of the aortic arch). The signal enters the spinal cord until reaching the NTS in the medulla oblongata, synapses with the RVLM and elicits an inhibitory response by decreasing sympathetic activity or increasing parasympathetic activity45. If a parasympathetic effect is required, the efferent output from the RVLM originates from the nucleus ambiguous and travels to the heart via the vagus nerve. The vagal fibers innervate the SA node, AV node, and the atrial myocardium, where it releases acetylcholine (ACh), which will activate the cholinergic receptors reducing the rate of firing of the SA node, the transmission of the AV node, and the atrial contractility. Additionally, the secretion of ACh by the vagus nerve inhibits the liberation of neurotransmitters by the sympathetic nerves, enhancing its inhibitory function46. 1.5 The cardiac cycle   The cardiac cycle is the period from the beginning of one heartbeat to the next heartbeat. In general terms, the cardiac cycle is divided into two broad phases, a filling phase called diastole, and an ejecting phase called systole. The cardiac cycle is initiated by an action potential (AP) which originates in the SA node in the posterior part of the RA. This AP travels through the atria in two ways: depolarizing the surrounding atrial muscle and by specialized conduction, fibers called intermodal pathways. At the same time that AP is being transmitted through the RA, the same AP is sent to the LA by the anterior interatrial band 21  which causes a simultaneous depolarization of both atria. After the transmission of the AP by the SA node, the AP reaches the AV node - which the only function is to transmit and the electrical signal from the SA node to the ventricles– and enters a layer of fibrous tissue that delays the pulse propagation and allows the atrial contraction to occur before the ventricular contraction. After passing the fibrous tissue, the AP continues to the AV bundle that then divides into the left and right bundle branches. These branches run parallel on each side of the ventricular septum below the endocardium and towards the apex, diving into smaller branches within the myocardium (Purkinje fibers) and go back to the base of the heart40. Given that the ramifications of the Purkinje fibers only penetrate one-third of the myocardium, the transmission of AP is completed by the same muscle fibers. Once the AP potential is fully propagated throughout the myocardium, the contraction of the ventricles takes place, and systole occurs40.  22   Figure 1.2 Wiggers diagram illustrating the correlation between the mechanical and electrical events during the cardiac cycle of the left ventricle.  The cardiac cycle diagram or Wiggers’ diagram (Figure 1.2) shows from top to bottom the electrical activity (ECG), aortic, atrial and ventricular pressure, ventricular volume and the heart sounds of the LV. During the systolic phase, the contraction of the myocardium causes an increase in pressure inside the ventricle closing the mitral valve (1st heart sound). When the pressure inside the ventricle exceeds the pressure in the arterial compartment, the aortic valve opens. After the ejection of blood from the ventricle, the diastolic phase begins with a rapid decrease of pressure and volume within the ventricle to a level below that in the systemic circulation causing the aortic valve to close (2nd heart sound). Subsequently, the pressure inside the ventricle continues to decrease until a pressure gradient is created between the atria and the ventricle. Due to the pressure gradient, the 23  atrioventricular valves reopen passively filling up to ~80% of the total diastolic volume with atrial contraction pumping the remaining blood to complete the diastolic filling. Once the diastolic phase has ended, another AP from the SA node is propagated through the ventricles repeating the cycle.  1.6 The pressure-volume relationship  The phenomena occurring inside the chambers of the heart during the cardiac cycle are variations in pressure and volume. The changes of pressure create pressure gradients from which the blood can move from one compartment to the other. These pressures are generated by the filling/emptying and the contraction/relaxation of the heart. An efficient and informative way to graphically represent these pressure changes are the “pressure-volume loops” (PV loops). The following section will focus on PV loops of the LV.   Figure 1.3 a) Pressure-volume loop, b) Time-pressure graph representing changes in pressure and volume during the cardiac cycle. a) b) 24   The direction of the loop is followed in a counter-clockwise direction, starting from the bottom right corner (Figure 1.3a). At the beginning of the cardiac cycle, the depolarization of the ventricle causes the myocardium to contract with a rise in pressure that causes the mitral valve to close without changes in volume, this phase in known as isovolumetric contraction (A). Subsequently, the pressure rises to a point where it overcomes the systemic pressure causing the aortic valve to open and ejecting blood into the aorta (B). Immediately after ejection, the pressure continues to increase until it reaches its highest point and then progressively decreases until the pressure generated by the ventricle gets to a point below the systemic pressure causing the aortic valve to close (end of systole). After the closure of the aortic valve, the diastolic phase of the cycle begins with an exponential pressure drop inside the LV at a constant volume, called isovolumetric relaxation (C). The pressure further continues to decrease until it reaches the pressure below the pressure in the LA opening the mitral valve (D) to fill the ventricle until the next systolic phase47. If the cardiac cycle is represented in a time-pressure graph (Figure 1.3b), the isovolumetric contraction (A) causes an increase in pressure (top section) with a constant volume. During ejection (B), the pressure continues to increase with a simultaneous decrease in volume within the ventricle (bottom section of Figure 2b) until the aortic valve closes (C). Then the mitral valve opens, and volume begins to fill the ventricle with a slight increase in pressure (D). From the PV loop, we can obtain several physiological parameters. For example, regarding pressures, the part of the cycle represented by A (Figure 1.3a) represents the pressure in the ventricle at the end of diastole. Following a counter-clockwise direction, the 25  highest peak of B represents the maximum pressure that represents systolic blood pressure and point C represents pressure at end-systole (Pes). Looking at the volume in the x-axis, SV can be obtained by subtracting end-diastolic volume (EDV) from end-systolic volume (ESV) (Figure 1.4). The SV among other factors will be influenced by changes in preload, afterload, and contractility (end-systolic pressure-volume relationship) that will be discussed next.   Figure 1.4 Stroke volume (SV) measurement in a pressure-volume loop by the difference between end-diastolic volume (EDV) and end-systolic volume (ESV).  1.7 Preload   Preload can be defined as the stretching of the myocardial fibers previous to contraction47, and it represents the tension at which the ventricle undergoes during the diastolic phase48. As previously discussed, preload is the basis of the Frank-Starling´s mechanism, as the increase in filling volume will generate an increase in pressure, this will stretch the myocardial fibers causing a change in length of the sarcomere, a better coupling of the contractile proteins, and an increase in SV (with contractility and afterload held constant). 26  The relationship between EDV and filling pressure (end-diastolic pressure-volume relationship) within the ventricle has important physiological consequences and varies depending on the compliance of the ventricular wall. For example, a heart with normal compliance presents an approximately curvilinear relationship49. Nevertheless, in the case of a stiff ventricle (e.g., hypertrophic cardiomyopathy), small increases in volume will generate exponential pressure increase without significant changes in the sarcomere length50. The ideal approach to measure preload would be by measuring the sarcomere length in vivo, unfortunately, this is not feasible. Instead, several methods are used to invasively assess preload such as measuring ventricular end-diastolic pressure, LA pressure, pulmonary venous pressure, and pulmonary capillary wedge pressure. However, in the clinical setting preload of the LV is estimated through the estimation of EDV by echocardiography51, 52.  1.8 Afterload   Afterload is defined as the ventricular wall tension before the ejection of blood by the ventricle. In other words, is the pressure load that the ventricle has to overcome to pump blood out to the peripheral circulation53. Afterload in the present document will refer to the resistance presented by the peripheral arterial system. In the LV, afterload is a parameter that reflects the TPR that opposes to blood flow. In the normal heart, an increase in afterload requires an increase in contractility to maintain SV and CO. On the contrary, a dysfunctional myocardium (e.g., congestive heart failure) where the cardiac functional reserve is limited, the heart is unable to increase contractility which leads to a lower SV54.  27  Measuring the wall stress within the ventricle is difficult to perform, but an estimation can be performed using the LaPlace relationship, where wall tension can be assessed by measuring the pressure, radius, and thickness of the ventricle with the formula: Wall tension= Pressure * radius / 2 * wall thickness.  In a laboratory setting, there are different measures of afterload including aortic pressure, TPR, arterial impedance, and peak wall stress. Each of these parameters assesses different aspects of the circulatory system whether by describing only the arterial properties or integrated with ventricular function55. 1.9 End-systolic pressure-volume relationship  A load-independent measure of cardiac contractility can be invasively obtained from a series of pressure-volume loops through the placement of a catheter into the LV, called the end-systolic pressure-volume relationship (ESPVR). These series or “family” of loops are obtained by changing loading conditions via occlusion of the vena cava that yield several end-systolic pressure-volume points (Figure 1.5). The slope formed by the family of ESPVR points at the top left corner is considered to be linear and intersects at a common point V0 in the volume axis with a slightly positive value, which represents the minimal amount of volume within the ventricle necessary to generate pressure. The slope originated from V0, and the ESPVR is called end-systolic elastance (Ees) and is a reflection of the intrinsic contractility of the myocardium56. Where the term elastance is defined as the change in pressure for a given volume57 and describes the “stiffness” or contractility of the myocardium. 28   Figure 1.5 End-systolic elastance (Ees) obtained from a series of pressure-volume loops under different preload conditions. The measurement of Ees to characterize the contractility of the ventricle has been used almost exclusively in animal studies, due to its invasive nature. One of the first to evaluate Ees was Suga and colleagues. In Suga’s experiments with excised canine hearts cautiously controlling preload and afterload, they demonstrated that the ventricular pressure-volume ratio at the end-systole is a parameter not affected by EDV58 or aortic pressure (afterload)59. He also showed that this measure of contractility greatly changes by infusing an inotropic agent such as norepinephrine60. Ees can determine the effectiveness of the heart as a pump and it has been used clinically during intervention procedures during circulatory shock to monitor response to inotropic agents61, to predict cardiac events in patients with negative stress echocardiography62 and to evaluate the aging effect on the cardiac response to exercise63. 1.10 End-systolic elastance   Besides the commonly used load-independent parameters of LV performance, preload recruitable stroke work (PRSW)64 and the ratio of LV maximal rate of pressure development to EDV (dP/dtmax-EDV)65, Ees is a more specific marker of systolic stiffness or contractility 29  of the myocardium at end-systole66, and it is determined by the passive and active properties of the myocardium67. To further clarify the two spectrums that Ees represents, stiffness and contractility, Young et al.68 conducted a study where they evaluated differences in resting Ees in a group of individuals with systolic dysfunction (non-ischemic dilated cardiomyopathy), hypertensive patients (cardiac stiffness) and marathon runners (highest contractility). Ees was the lowest in the systolic dysfunction group, reflecting a reduced contractile function. While there was no difference between the marathon runners (higher contractility) and hypertensive (stiff myocardium), highlighting that Ees can represent both myocardial conditions and that this parameter has to be put into context when interpreting ventricular elastance values. Ees is a crucial determinant of ventricular performance and most importantly its interaction with the vascular system, called ventricular-arterial coupling (VAC), will predict the ability to increase ventricular function during stress69. As previously discussed, Ees is obtained using a PV loop which implies measuring changes in pressure and volumes with a catheter within the ventricle 60. Due to the invasive nature of this procedure, the determination of ESPVR to obtain Ees is not practical in the clinical practice to monitor pharmacological therapy or exercise interventions. To address this limitation, Chen et al.70 validated a non-invasive approach to obtaining single-beat Ees (Ees(sb)). This approach involves measuring BP, and echocardiographic measures including Doppler-derived SV, ejection fraction (EF) and an estimated normalized ventricular elastance at end-diastole with the following formula:  Ees(sb) = [Pd - (ENd(est) * Ps * 0.9)]/(ENd(est) * SV)  30  In the formula, Ps and Pd are systolic and diastolic BP, and END(est) is the normalized elastance curve obtained from a regression model based on invasive PV loop assessment and using 7 term polynomial function including EF, SPB and DBP, and the ratio of pre-ejection period (PEP) to total systolic period (TSP)70. This single-beat approach has been used in clinical trials that evaluate the impact of blood pressure-lowering drugs on arterial and LV stiffness to improve cardiac mechanical efficiency71 and in cardiac rehabilitation programs to improve systolic function72. 1.11 Ventricular stiffness index  Another approach to assess the contractile function of the LV is the ratio of peak blood flow velocity/time to peak velocity ratio (ventricular stiffness index) in the LV outflow tract measured by Doppler. This non-invasive marker of ventricular contractility has been compared to the invasive gold-standard Ees obtained from PV loops and has shown to be preload independent and sensitive at detecting changes in contractility in the presence of cardiac disease68, 73. One of the first studies to assess systolic function through Doppler indices and compare them against invasive parameters was conducted by Wallmeyer et al.74. Using six dogs, they compared invasive methods including maximal blood flow in the descending aorta (Qmax), maximal rate change of blood flow (dQ/dt max), and maximal rate of rise of pressure of the LV during isovolumetric contraction (dP/dtmax) with Doppler-derived peak blood velocity (PBV), time-to-peak velocity (TTP) and their ratio under different inotropic states. The PV and the ventricular stiffness index (VSI), detected changes in ventricular contractility with dopamine and propanolol infusion and showed to be highly correlated with the invasive parameters74. In a more recent study,  Bauer et al.73 compared in sheep, VSI and 31  invasive Ees under different loading conditions. They observed that VSI was not affected by loading conditions and that it was not significantly different from Ees at detecting inotropic changes of the LV73. In humans, VSI was able to detect differences in ventricular inotropic states between individuals with high systolic function (marathon runners), stiff ventricles (hypertensive), and decreased contractility (non-ischemic dilated cardiomyopathy)68. 1.12 Arterial elastance  The gold standard to evaluate the characteristics of the arterial tree and with this, the resistance that the ventricle has to overcome to eject blood (afterload) is based on a three-element Windkessel model proposed by Westerhof et al.75. This model describes the association between the pulse wave, the interaction of SV, and the properties of the arterial system and includes the resistance to flow in the proximal aorta (characteristic impedance), arteriolar resistance (TPR) and large artery ratio of volume change and pressure (total arterial compliance)76. This analysis is performed by a Fourier analysis of aortic impedance (forces that resist ventricular ejection) measuring the flow and resistance in the proximal aorta77. The Fourier analysis involves a sophisticated analysis of flow fluctuations, as detailed by Kelly and colleagues78. On the other hand, EA is a commonly used parameter that also describes the general vascular loading conditions on the LV. EA integrates the steady and pulsatile components of arterial circulation and is easily determined by the intersection of Pes with EDV of the PV loop (Figure 1.6). Sunagawa et al.79 proposed a formula to approximate the value of EA by knowing the resistance, compliance, impedance, systolic and diastolic time intervals from the arterial impedance approach with the following formula:  32  Ea (Z) = RT / [ ts+ Ʈ (1-etd/Ʈ) ] Where RT is systemic vascular resistance, ts and td are systolic and diastolic time, respectively. Ʈ represents tau or time decay constant. With this equation, the information obtained from a 3-element Windkessel model can be expressed as mmHg/ml, just as the elastance in the LV. By using the same units to characterize the arterial and ventricular properties, it allows us to analyze the interaction or coupling of these systems by using the VAC ratio (EA/Ees). In a PV loop, the VAC ratio is obtained with the intersection between EA and Ees (Figure 1.6) and allows the evaluation of cardiac mechanical efficiency, which will be discussed over the next sections.   Figure 1.6 The intercept of end-systolic elastance (Ees) and arterial elastance (EA) representing ventricular-arterial coupling (VAC).   EA as a measure of afterload is mainly affected by three components including resistance, compliance, and HR. However, a study conducted by Segers et al.77 using a heart-interaction model studied the effects of peripheral resistance and arterial compliance on EA that cover the pathophysiological vascular conditions in humans. They observed that EA is 33  three times more sensitive to changes TPR compared to compliance (arterial stiffness). These findings are supported by a study conducted by Chemla and colleagues80, where they observed in hypertensive and normotensive individuals that EA is more susceptible to be affected by resistance, rather than by compliance.  1.13 Ventricular-arterial coupling   1.13.1 Ventricular-arterial coupling as a parameter of cardiac performance   The interactions between the LV and the arterial system are critical determinants of cardiovascular performance. For this, the ratio of EA/Ees or VAC has shown to be a reliable index to characterize the mechanical efficiency of the heart, LV energetics, and performance of the ventricle in situations of remodeling, fibrosis, and heart failure with reduced or preserved EF81. Mechanical efficiency per se is the maximal amount of energy transferred from its source without a significant loss of energy in the process82. In terms of cardiovascular function, mechanical efficiency is the maximal amount of blood transferred from the ventricle to the arterial circulation without losing significant energy overcoming the viscoelastic properties of arterial system83, this is accomplished in a “coupled” VAC state when both elastances are equalized (EA/Ees= 1)67, 69, 84. The VAC ratio can also be used to evaluate the LV energetics - minimal energy consumption to maintain cardiac output - which is accomplished when EA is half the value of Ees, resulting in a VAC ratio of 0.5. Both the mechanical efficiency and LV energetics can be graphically determined with two areas in the PV loop, the stroke work (SW) and the potential energy (PE) (Figure 1.7). The energy produced by the ventricle and transferred into the arterial system is assessed by the enclosed area that represents SW, and the triangle formed below represents the PE and reflects the energy accumulated and stored in the ventricle during systole. The area composed 34  by the SW and PE constitutes the pressure-volume area (PVA) which correlates with myocardial oxygen consumption per beat and thus, the total mechanical energy generated by the LV. Given that PVA represents the energy generated by the ventricle, the energy efficiency can be calculated as the ratio of SW/PVA.  Figure 1.7 Stroke work (SW) and the potential energy (PE) in a pressure-volume loop.  Different clinical scenarios can cause an unbalance in the VAC ratio (EA/Ees >1) creating a state that is referred to as “ventricular-arterial uncoupling.” This uncoupled state can be caused by either a decrease of Ees (heart failure: ischemic, myocardiopathy, septic shock, etc.) or an increase in EA (acute blood pressure increases, chronic pulmonary hypertension, increased vascular resistance). When the cardiovascular system is stressed as in exercise, the greater increase in contractility (>Ees) relative to the increase in vascular load (EA) cause a decrease in VAC ratio, this represents a higher mechanical efficiency necessary to supply the metabolic demands.    35  1.14 Cardiovascular pathophysiology in SCI  1.14.1 Introduction  Spinal cord injury (SCI) is a chronic condition that significantly impacts the quality of life and causes high healthcare costs such as rehabilitation, treatment of complications in the chronic phase, and loss of productivity85. In Canada, an estimated economic burden per individual in their lifetime is calculated to be $3.0 million for tetraplegia and $1.5 million for paraplegia86 with an estimated prevalence of 85,566 individuals in 2012 (51% traumatic SCI)87. In British Columbia, it is estimated that the incidence is 35.7 per million individuals88. Annually, nationwide $2.67 billion is spent, including long-term complications such as respiratory infections, bladder and bowel dysfunction, and pressure ulcers86. Given the therapeutic advances in treating these complications, life expectancy has increased and so to have CV complications, which are now one of the leading causes of death in this population89. It has been reported that individuals with SCI exhibit 2.7 fold increase for heart disease and a 3.7 increase for stroke90. A necessary approach to try to decrease risk factors associated with CV disease in these individuals is the implementation of an appropriate physical activity program.  1.14.2 Blood pressure dysregulation   The regulation of BP is influenced by many factors including neural, hormonal, renal and local tissue control acting in the short or long-term control of BP 91. During abrupt changes in BP three main mechanisms restore BP levels, including arterial baroreceptors, chemoreceptors and hypoxic stimulus in the vasomotor centers in the medulla. Rapid regulation of these mechanisms is carried out by increasing or decreasing the firing rate of 36  sympathetic nerve fibers to stimulate or inhibit cardiac function and vascular resistance, respectively91. Due to the segmental innervation of the sympathetic system, the level of injury (cervical, thoracic or lumbar) will determine the magnitude of supraspinal sympathetic control. For example, in cervical injuries, the lesion will disrupt the central sympathetic input to the organs and peripheral blood vessels. This will be reflected as low resting arterial blood pressure, inability to increase BP during exercise, unstable BP with postural changes (orthostatic hypotension) and sudden hypertensive bouts also known as autonomic dysreflexia (AD)27.  The exact mechanism of orthostatic hypotension (OH) in SCI is unclear. However, several factors could influence postural change intolerance besides SNS dysfunction92. These factors include a reduced baroreflex sensitivity in both tetra- and paraplegic individuals observed in studies with neck suction or pressure to stimulate carotid baroreceptors93, 94. The loss of supraspinal control over the adrenal glands results in significantly lower catecholamine levels that further blunt a compensatory vasoconstrictor response95. When changing to an upright position, the lack of skeletal pump due to leg paralysis significantly decreases venous return to the heart resulting in lower SV, CO and BP96. Additionally, the amount of venous return is also affected by the lower plasma volume in SCI secondary to hyponatremia97. Hyponatremia in SCI could be explained by reduced water and dietary salt intake and increased anti-diuretic hormone activity that leads to lower plasma volume, predisposing to more severe and frequent episodes of OH98. Lastly, the combination of reduced blood volume, altered sympathetic function, and reduced muscle or tissue pressure might lead to cardiovascular deconditioning commonly seen in prolonged bed-rest or inactivity, as in SCI, negatively affecting the normal CV response to postural changes99. 37  Aside from OH, individuals with SCI – especially high thoracic or cervical injury – may present frequent life threatening episodes of high and uncontrolled systolic (up to 300mmHg) and diastolic BP (>120mmHg)100 known as autonomic dysreflexia (AD)92. Episodes of AD can manifest as throbbing headache, bradycardia, and upper body flushing or go asymptomatic and simply manifest as sweating and piloerection101. These AD episodes can be elicited noxious (e.g pressure ulcers) or non-noxious stimuli (e.g. tight clothing)102. There are several mechanisms that might explain this response including vascular α-adrenoceptors hyperresponsiveness103 (upregulation of α-receptors or abnormal presynaptic reuptake of NE), abnormal baroreflex response, or loss of tonic bulbospinal inhibition102; however, the exact mechanism is yet to be clarified. 1.14.3 Cardiac structure in SCI  The heart has been recognized to have plastic properties or adaptation capability to increased stress, whether induced by increased or decreased volume or pressures104. In the case of decreased loading conditions, such as prolonged bed rest105 and spaceflights106, the heart exhibits a decrease in dimensions reflected as smaller LV mass, decreased wall thickness and lower EDV. In the case of SCI, also a state of cardiac unloading (decreased venous return and blood volume), the chronic unloading conditions and the loss of trophic stimulation by the sympathetic system107 has shown to cause cardiac atrophy in both human108 and animal studies109, 110. Several studies have reported structural changes, especially in cervical injuries, including a decrease in EDV, LV internal diameter at end-diastole (LVIDd), and LV mass, along with concentric remodeling111, 112. Additionally, it has been reported in animals with high-level SCI the presence of cardiomyocyte atrophy 38  (decreased length and width), a reduced number of contractile units per cell and upregulation of ECM degrading enzymes109.  1.14.4 Systolic function in SCI  Acutely after high-level SCI, individuals commonly present electrical conduction abnormalities such as brady- or tachyarrhythmias, low resting HR (unopposed parasympathetic stimulation), reduced SV, CO and MAP (due to systemic vasodilation)113. Chronically, the risk of cardiac arrhythmias decreases over time, but the functional impairments remain. The impact of SCI on the heart’s function is dependent on the level of injury, sparing individuals with paraplegia114 and severely affecting individuals with complete or incomplete (ASIA A-B) cervical injuries in terms of cardiac atrophy and impaired systolic function111, 112, 114, 115. In animal studies with chronic SCI, it has been reported that SCI compromises the pressure-generating capacity of the LV (dP/dtmax), load-independent parameters of contractility, and volumetric indices109, 116. In clinical studies, it is reported that tetraplegics present lower SV, CO and slightly reduced111, 112 or preserved EF117 compared to AB, but still above the 55% EF threshold118.  1.14.5 Diastolic function in SCI   Finding in studies investigating diastolic function in cervical SCI have not been consistent. The studies conducted by Eysman et al.115, Groot et al.117, Matos-Souza et al.119 and Schreiber et al.120 did not find a difference in at early-to-late LV diastolic filling ratio (E/A) compared to AB. However, both Mato-Souza et al., and Schreiber et al., did report impaired diastolic function with higher mitral early filling peak velocity (E) and annular early diastolic tissue velocity (Em) ratio (E/Em), primarily due to lower Em119, 120. Diastolic 39  function in SCI could be affected by the level of physical activity112, 121, lower blood volume122, and increased expression of the peptides of the renin-angiotensin system (RAS)120 that could induce ventricular stiffness. In animal models, it has been reported that the chronic and frequent incidents of sudden BP rises (autonomic dysreflexia), commonly seen in SCI, negatively affect diastolic function 123 and at the cellular level in SCI animals, causes an upregulation of matrix metalloproteinase 9 (MMP9) and its inhibitor, tissue inhibitor of metalloproteinase 1 (TIMP1), which contribute to the degradation of extracellular matrix 109 and that have been associated with diastolic dysfunction124. 1.14.6 Vascular structure in SCI  Acutely after SCI, the effect of lost supraspinal neural control and reduced physical activity125 have a deleterious effect on arterial structure showing an inward remodeling pattern with decreased vessel diameter and an increased wall thickness of conduit arteries, associated with muscle atrophy and lower metabolic demand126. De Groot et al. (2006) reported in high and low-level SCI, a decrease in the diameter of the superficial and the common femoral artery (CFA) as early as 3 weeks post-injury, but no change in upper body arteries (carotid and brachial arteries)127. Chronically, the absolute diameter of femoral arteries in SCI individuals remains 37% lower compared to AB128, 129, but once adjusted for leg volume the diameter it is not significantly different from AB129. Regarding the upper body vasculature, it has been reported a preserved130 or even greater128 brachial artery diameter and similar carotid diameter compared to AB131.  Studies evaluating carotid wall thickness in both tetraplegic and paraplegic have found a higher carotid intima-media thickness compared to non-injured individuals131, 132. Thijssen and colleagues (2012) in a follow-up study of 24 weeks post-injury, observed 40  significant increases in wall thickness over time in the carotid and CFA 133. Along with a thicker arterial wall in SCI, it has been found that SCI increases aortic stiffness measured by carotid-femoral pulse wave velocity134, 135, regardless of the level of physical activity135. 1.14.7 Vascular function in SCI  Studies in humans have shown that SCI has little effect on upper body endothelial function. For instance, NO dependent endothelial function assessed by FMD has been reported to be normal in brachial arteries136. However, studies looking at cerebrovascular function in humans have reported an altered dynamic cerebral regulation, vascular reactivity, and neurovascular uncoupling after high-level SCI. Furthermore, animal studies have found, in large cerebral arteries, profibrotic changes, increased stiffness and inward remodeling, implying SCI alters the vasculature above the level of injury137. Similarly, an experimental SCI rodent study showed a profibrotic phenotype (increased collagen I and tissue transforming growth factor β), endothelial dysfunction, and inward remodeling in the femoral artery138, contrary to the preserved or even enhanced endothelial function reported in the superficial femoral artery (SFA) in humans after SCI136.  In chronic SCI, noxious and non-noxious visceral (bladder or bowel distension) or somatic stimuli below the level of injury can trigger an exaggerated sympathetic response manifested as a sudden increase in SBP (20 mmHg) with or without reflex bradycardia and flushing above the level of injury, among other symptoms27. These repetitive BP increases have been related to a decrease in the number of elastic proteins in the vascular wall of the femoral artery in animals139 and might be one of the factors associated with arterial dysfunction in SCI. In animal studies, after the infusion of phenylephrine (adrenergic agonist) animals with SCI present a significantly greater vasopressor response compared to 41  controls (Figure 1.8a). Furthermore, using colorectal distension to induce repetitive increase bouts of BP to mimic AD, it was reported that animals that underwent colorectal distention (SCI-CRD) exhibited greater vasoconstrictor response to phenylephrine infusion (Figure 1.8b) compared to those without the repetitive stimulus. The increased pressor response might be secondary to a lower level of circulating catecholamines (due to loss of sympathetic activity and denervation of adrenal glands) after SCI, a higher adrenoceptor sensitivity (upregulation of α-adrenoceptors)140 or an increased VSMC response.  Figure 1.8 a) Increased vasoconstrictor response of femoral artery rings in SCI rats (open dots) after increasing phenylephrine infusion compared to controls (closed dots) Source: Alan et al.141. b) Enhanced vasoconstrictor response after phenylephrine infusion (PE) in rats that underwent repetitive colorectal distension (SCI-CRD) to induce autonomic dysreflexia, compared to SCI controls (SCI). Source: Lee et a.140.  1.14.8 Stroke volume, cardiac output, and heart rate during exercise in SCI  Due to loss of motor function of skeletal muscle and decreased sympathetic input to peripheral vasculature, there is a marked reduction in skeletal pump function142, increased compliance of the venous compartment, lack of redistribution from the splanchnic area to active muscles143, and venous pooling in the lower limbs144, 145. This retention of blood in the a) b) 42  lower limbs and splanchnic areas will decrease venous return to the right side of the heart with reduced EDV, and according to the Frank-Starling mechanism, will present a lower SV, with a further compensation of HR to maintain CO. Studies examining changes in SV  with upper body exercise showed that cervical SCI individuals are able to slightly increase their CO during exercise based on significant changes in HR, with no changes of  SV from rest to maximal exercise intensity146. Meanwhile, studies assessing CV changes in SCI with only passive leg cycling to increase venous return have shown that SV can increase up to 29% compared to rest, with no changes in HR or BP147, 148. Using electrically stimulated leg contractions in tetra- and paraplegics, Flemming et al.,149 reported in both SCI groups, a significant increase in SV and CO compared to rest and a slight increase but delayed HR response. The dependency on HR to increase CO is associated with the level of injury, as demonstrated in a study by Schmid et al. in SCI95. In their study, they reported that the higher the level of injury, the steeper the relationship slope between HR and oxygen consumption during exercise, as shown in Figure 1.9. In individuals with a lesion below T6, there is an intact brainstem to the heart; however, the control of the splanchnic and renal circulation might still be compromised affecting blood flow redistribution and the neurohormonal control during exercise32.  43   Figure 1.9 Relationship between HR and oxygen consumption (VO2) in able-bodied (AB), low thoracic paraplegic (MLPara), high thoracic paraplegic (HLPara) and tetraplegic (Tetra). Source: Schmid et al. 199895.  1.14.9 Cardiac responses to exercise in SCI  In highly trained AB endurance athletes it is common to find physiological cardiac remodeling characterized by hypertrophy150 and low resting HR32; however, it does not seem to be the case in SCI athletes. West et al.112  showed that tetraplegic athletes still present a reduction in LV mass, LV end-diastolic diameter and volume with no differences regarding diastolic function. In comparison to sedentary tetraplegics, De Rossi et al reported a better diastolic function in SCI athletes, by means of increased by early mitral flow velocity (e’) an increased early diastolic function and a reduced (improved) early mitral flow velocity to e’ ratio (E/e’). In addition, most clinical studies report a decrease in estimated LV mass in athletes with high-level SCI112, 151. Unfortunately, the cross-sectional nature of studies looking at the effect of long-term exercise on cardiac remodeling in SCI does not allow to infer a its direct effect on the heart. Nevertheless, a study in rodents showed that early or later 44  hindlimb exercise training could help reduce the reduction in cardiac volumes after SCI, but such increases in volume are not considered to be associated with improved contractile function152.  1.14.10 Blood pressure during exercise in SCI  The normal BP response during cardiometabolic stress like exercise is mediated by changes in TPR and CO. These responses are regulated by the descending sympathoexcitatory input to the peripheral vasculature and the contractility of the heart. In SCI, the lack of cardiovascular response depends on the level and completeness of the injury. For instance, in tetraplegics, studies have shown significantly lower resting values of SBP, DBP, and HR as compared to high thoracic or low thoracic paraplegics and that during arm crank exercise they are incapable of increasing their BP and even drop their BP after maximal exercise153-155. Whilst, paraplegic individuals with low-level thoracic injuries (T7 or below) show a significant increase in the MAP, possibly due to a better blood redistribution from the splanchnic area to the active muscles and greater peripheral vasoconstriction (greater sympathetic input) to increase preload, compared to tetraplegic individuals. Furthermore, studies looking at catecholamine levels during exercise with different levels of injury have shown that cervical SCI has the lowest resting levels of catecholamines and present no significant increase after maximal exercise, and the level of injury is negatively associated with the catecholamine levels95, 156. 1.14.11 Effect of exercise on vascular structure and function in SCI  SCI impairs the normal cardiocirculatory response to exercise and profoundly decreases exercise capacity. There are several exercise modalities for individuals with SCI 45  including arm crank ergometry (ACE), functional electric stimulation (FES), hybrid (ACE + FES), passive lower-limb cycling, bodyweight-assisted treadmill training, etc. However, the effects of ACE will be discussed since it is a common, simple, and widely reported exercise method among SCI and is the primary exercise modality of wheelchair rugby athletes (besides resistance training). Little-to-no evidence is available to support that ACE improves arterial function. For the acute vascular changes after exercise, one study looked at changes in blood flow in the femoral artery in SCI and AB. The study reported no changes in diameter, SR or SBP necessary to cause mechanical stimuli to improve endothelial function157. For the chronic effects, there is only one published randomized controlled trial that studied the impact of 16 weeks of aerobic and resistance training on the vasculature in SCI. They found that the intervention group exhibited a slightly improved carotid distensibility with a concomitant distensibility reduction in the control group158. Interestingly, both of the previously mentioned studies reported that some SCI individuals showed a positive vascular response in acute and chronic exercise interventions. However, the reason why some SCI individuals are “responders” is not clear. Although, the mechanical stimuli to improve vascular function in SCI in absent, upper body exercise could improve it by reduction of oxidative stress159, inflammatory makers160-162, and insulin resistance162-164 – which are related to arterial stiffness165 – which could help attenuate an accelerated vascular dysfunction in SCI166.    46  1.15 Aims and hypotheses 1.15 Aims  i. To measure and compare EA, Ees(sb) and VSI and their ratio EA/Ees(sb) and EA/VSI, at rest in SCI-a, SCI-na and AB controls. ii.  To validate the use of VSI in SCI using an experimental animal model by correlating VSI with invasive Ees, PRSW and dP/dtmax-EDV. 1.16 Hypotheses  - Non-athletic individuals with chronic cervical SCI will present a higher VAC ratio compared to wheelchair rugby athletes with cervical SCI, while AB will present the lowest VAC of the three groups. - VSI will exhibit a high correlation with invasive Ees, PRSW and dP/dt-EDV.         47  Chapter 2: Non-invasive ventricular-arterial couplings in humans and animals with SCI.  2.1 Brief introduction  CVD is the 2nd leading cause of death in Canada. It is estimated that 1 in 12 Canadians over 20 years old have a diagnosed heart disease167, 168. In individuals with SCI, there is a three and four-fold increased risk for heart disease and stroke, respectively90. A common predictor for adverse CV outcomes is EF, but due to changes in cardiac volumes associated with SCI, EF has failed to show108 the presence of systolic dysfunction post-SCI. In rodents with SCI, where direct LV catheterization can be performed there is clear evidence of systolic dysfunction in animals with SCI109, 169, 170. A potentially more comprehensive assessment of the function of the CV system that can be performed in humans is the estimation of the contractile capacity of the heart (i.e. end-systolic elastance [Ees]), the resistance to stroke flow by the arterial system (i.e. arterial elastance [EA]), and their interaction (EA/Ees), called ventricular-arterial coupling (VAC). This interaction provides information on the mechanical and energetic efficiency of the CV system171. An “uncoupled” or high  (VAC>1) EA/Ees ratio is associated with lower cardiac reserve during exercise63, and risk of all-cause mortality, cardiac transplantation or hospitalization in patients heart failure (HF)172. After high-level SCI, animal studies have shown an increased VAC ratio due to a reduction in cardiac contractility and increased resistance to flow109, 169; however, no studies have investigated VAC in humans. Therefore, the aim of this investigation was to evaluate VAC in non-physically active individuals with chronic SCI (SCI-na) and compare VAC against able-bodied individuals. The impact of chronic intense exercise on VAC was also investigated by assessing elite Paralympic athletes with chronic cervical SCI (SCI-a) that 48  take part in competitive wheelchair rugby. Moreover, to support the validity of human findings, we retrospectively validated our non-invasive index of LV contractility by comparing this index against invasively determined ESPVR, maximal rate of pressure change normalized to cardiac volumes (dP/dtmax-EDV) and preload recruitable stroke work (PRWS) in rats with SCI.               49  2.2 Methods  2.2.1 Study design  This study was performed in two stages: a cross-sectional comparison of arterial and cardiac parameters in humans, and a retrospective validation of the VSI measure in a rodent model. The evaluation of human participants was performed in one visit for the SCI-na and AB groups. During this visit, a brief questionnaire about physical activity (Leisure Time Physical Activity Questionnaire for People with Spinal Cord Injury), anthropometry, and medical conditions were applied. Hemodynamic evaluation and arterial tonometry were performed by a trained examiner. Likewise, ultrasound was performed by a trained sonographer throughout the study. Examinations in the laboratory were carried out in a controlled environment and fixed room temperature. For the SCI-a, the same assessments were performed during the Wheelchair Rugby national tryouts at the Richmond Olympic Oval. All procedures were identical across all 3 groups. For the animal study, ultrasound and PV loop analysis were performed on Wistar rats from a previously completed study. We included data from sham and SCI rats with a complete transection at the 3rd thoracic segment. In these rats, ultrasound was performed pre- and 9 weeks post-SCI and invasive LV catheterization was performed at study termination (9 weeks post injury). 2.2.2 Study participants and experimental units  Human participants. A sample of 44 individuals was recruited from Vancouver and neighboring areas. The participants were recruited from the Yuel Family Physical Activity Research Centre (PARC) in the Blusson Spinal Cord Centre, local rehabilitation centers, and community SCI networks. Athletes with cervical SCI were recruited from the National 50  Rugby Team tryouts and AB controls were recruited locally from within our research center. The SCI individuals consisted of males and females aged 18-60 with motor complete (AIS A-B) chronic (>1yr post-SCI) cervical SCI, who do not take part competitive sport (n=13; SCI-na) and elite level wheelchair rugby athletes (n=15; SCI-a). AB individuals consisted of males and females aged 18-60 (n=15). All of the participants were free from cardiovascular, metabolic, respiratory, or renal disease. All participants were instructed to refrain from ingesting alcoholic or caffeinated beverages and strenuous exercise 24 hours prior to the study and to be at least 2 hr postprandial. All testing was completed on a single visit. Animals. The data of ten adult Wistar rats were retrospectively analyzed with 5 animals per group (SCI and sham). Animals in the SCI group were surgically injured with a transection at the third spinal level (T3 level) to produce a loss of descending sympathetic input to the heart and vasculature. Sham animals underwent laminectomy at the same level, but without injury to the spinal cord. Both animal groups received the same post-operative management with weight-adjusted enrofloxacin (10 mg/kg) and buprenorphine (0.02 mg/kg) for 3 days post-surgery. Ultrasound was performed pre-SCI and 9 weeks post-SCI. Invasive parameters of cardiac function were assessed 9 weeks post-injury. Details of the surgical procedure as well as the pre- and post-surgical care have been reported elsewhere173. 2.2.3 Specific methodology (Humans)  2.2.3.1 Hemodynamic assessment  Brachial BP was measured in a supine position after a 10 min rest with an automatic sphygmomanometer (Carescape™ V100; GE Medical Systems, Milwaukee, WI, USA). Three measurements were performed, using the average of the last two for the analysis. 51  Central SBP was estimated obtaining pulse wave contour on the right carotid artery with a hand-held high-fidelity tonometer (Model SPT-301, Millar Instruments Inc., Houston, TX, USA). The DBP and MAP were used to calibrate and determine central SBP, as recommended by current guidelines174. 2.2.3.2 Vascular structure and function   Vascular structure and function were assessed by ultrasound of the right common carotid artery. The carotid structural evaluation was performed using high-resolution B-mode ultrasound (Vivid 7; GE Medical, Horton, Norway) with a 7 MHz phased array linear transducer. The vascular characteristics were assessed by 10- second video clips in a horizontal projection of the vascular wall. All images and videoclips were stored for offline analysis with EchoPac Software (GE healthcare). The vascular structure was evaluated by determining external and internal diameter in diastole, lumen diameter, and intima-media thickness (IMT). The external diameter was measured on the arterial outer edges from a longitudinal view. Lumen diameter was measured as the distance between the anterior and posterior intima lumen echoes175. The IMT was measured on the far wall with a minimum of 10 mm length of the arterial segment following the Mannheim Advisory Board Consensus recommendations176. Subsequently, IMT was measured with a semi-automatic edge-detection software and an average of six measurements in diastole were used for analysis.   Vascular function was assessed by estimating local parameters of wall stiffness and shear rate.  The arterial wall stiffness was evaluated with β stiffness index, distensibility coefficient (DC), local pulse wave velocity (PWV), elastic modulus (Einc), and compliance 52  coefficient (CC). The previously mentioned parameters were calculated with the following equations177:  β stiffness index β=ln ( PsPd )(Ds - Dd)/ Dd Where Ps is systolic BP, Pd is diastolic BP, Ds and Dd are arterial diameters at systole and diastole, respectively. β represent the stiffness of the arterial wall independent of pressure changes178. Distensibility coefficient DC=(2∆D ∙D+∆D2) (∆P ∙ D2) Where ∆D is diastolic-to-systolic diameter, D is diastolic diameter and ∆P is pulse pressure. DC reflects the relative diameter change by a given pressure increase177. Local pulse wave velocity (PWV) Local PWV= √ 1 / (ρ∙DC)   Where 𝜌𝜌 is density of blood assumed to be 1050 kg/m3 179 and reflects an estimation of the local PWV as arterial stiffness parameter based on DC calculation179,. Einc reflects the pressure required for a 100% theoretical stretch from resting diameter and provides information about the elasticity of the wall material independent of its geometry180. Incremental elastic modulus 𝐸𝐸𝑖𝑖𝑖𝑖𝑖𝑖 = 3 x (1 + 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝑊𝑊𝐿𝐿𝐿𝐿𝐿𝐿)DC  53  Where LCSA represents a longitudinal cross-sectional area and WCSA represents wall cross-sectional area180.  LCS and WCSA were evaluated with the following formulas:  Longitudinal cross-sectional area 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 = 𝜋𝜋 ∙ 𝐸𝐸𝐸𝐸24   Wall cross-sectional area WCSA=π ∙ (ED2- Dd2)4 Where ED is the external diameter.  Compliance coefficient CC=π(2D ∙ ∆D+∆D2) 4∆P Where π is Pi (3.14159), CC reflects the absolute diameter change for a given pressure increase177. In addition, Duplex ultrasound was used to measure peak blood velocity and arterial diameter continuously. The volume sample was located in the carotid longitudinal axis at a 60-degree angle. This technique allowed the determination of the shear rate (SR) using the following equation181: Shear rate SR = 8 ∙ ud Where u is the average velocity and d represents vessel diameter.    54  2.2.3.3 Cardiac structure and function  Participants were evaluated in a supine position on a specialized echocardiography bed with a cushion under the right upper back to slightly tilt the participant to the left and optimize image acquisition. Following 5 minutes of rest, heart ultrasound was performed with participants in a left lateral decubitus position. Images were obtained at end-expiration using a 1.5-4 MHz phased-array transducer with a commercially available ultrasound (Vivid 7; GE Medical, Horton, Norway) and stored for offline analysis (EchoPAC, GE Healthcare). To estimate basic structural and volumetric characteristics, and non-invasive VAC, we obtained images from two-dimensional (2D) 4-chamber and 2-chamber view, para-sternal long-axis view, and aortic pulsed-wave Doppler from an apical 5-chamber view.  2.2.3.4 Non-invasive VAC ratio  Single-beat Ees. The VAC using single-beat Ees (Ees(sb)) was calculated as: VAC(sb) = EA(Doppl) / Ees(sb) EA was estimated with the formula EA(Doppl) = (SBP x 0.9) / Doppler derived stroke volume (SVDoppl)60. Calculation of single-beat Ees was performed as proposed by Chen et al.70 with the following formula:  Ees(sb) = (DBP – [End(est) x SBP x 0.9)] / End(est) x SVDoppl Where DBP and SBP are brachial diastolic and systolic BP, SVDoppl is calculated from Doppler, and End(Est) is the estimated normalized ventricular elastance at the onset of ejection and calculated with the following formula: End(est) = 0.0275 − 0.165 × EF + 0.3656 × (DBP∕SBP × 0.9) + 0.515 × End (avg)  55  Where EF is obtained from Simpson´s biplane method of disks summation and End(avg) was calculated from a 7-level polynomial formula: End (avg) = 0.35695 − 7.2266 × tNd + 74.249 × tNd2 − 307.39 × tNd3 + 684.54 × tNd4 –856.92 × tNd5 + 571.95 × tNd6 − 159.1 × tNd7   tNd represents the ratio of pre-ejection to total systolic period measured at the left ventricular outflow tract (LVOT).  VAC calculated with VSI. As a novel surrogate for invasive Ees, VSI was calculated measuring peak blood velocity and time-to-peak by pulsed-wave Doppler from the LVOT with the formula68: VSI = Peak blood velocity / Time-to-peak To calculate VAC in humans, the following formula was used as reported in a previous clinical study68: VAC= 10 x EA(Simp)/VSI. Where EA(Simp) was calculated using the same formula as previously mentioned but using Simpson’s biplane method to estimate SV following current guidelines118. 2.2.4 Specific methodology (Rodents)  2.2.4.1 Cardiac ultrasound  Animals were anesthetized with isoflurane 4% for induction and isoflurane 1.5-2% for maintenance during heart ultrasound. Basic LV structure and function were obtained with an ultra-high frequency transducer (22-55 MHz) with commercially available ultrasound 56  (VEVO 3100, Fujifilm VisualSonics, Toronto, ON). Given the technical difficulty to obtain an optimal Doppler signal from LVOT in rats, VSI was calculated from the pulmonary trunk. Five to six consecutive end-expiration Doppler traces were used for analysis. PBV was measured at the maximal point of blood flow velocity, and TTP was measured as the time from the onset of flow to the peak of the systolic velocity trace. Rodent HR was obtained from the ECG signal integrated into the ultrasound system. The analysis of the echocardiographic parameters was performed blinded with commercially available software (VEVO 3100 Imaging platform).  2.2.4.2 Invasive parameters of systolic function in rodents  Following established procedures173, LV function was assessed with a closed-chest approach under urethane anesthesia and by insertion of a pressure-volume (PV) admittance catheter (Transonic SciSense, London, ON) through the right common carotid artery. The catheter was advanced to the LV until obtaining an optimal and stable PV signal according to established guidelines173. The volume signal was corrected to echocardiography-derived volumes. Following 10-min of hemodynamic stabilization we performed a laparotomy followed by compression of the inferior vena cava (IVC) with a cotton-tipped applicator to reduce preload and obtain a series of PV loops that enables calculation of Ees. Data were stored for offline analysis and analyzed blinded using LabChart 8 (ADInstruments, Inc., Colorado Springs, CO). Ees was determined as the slope of a series of ESPVR points during the IVC occlusion and EA was determined at baseline as the intersection between ESPVR and EDV. The VAC ratio was calculated as EA/ Ees. PRSW was estimated by the slope obtained from plotting SW and EDV from a series of PV loops obtained during the IVC occlusion. Likewise, dP/dtmax-EDV was calculated as the slope of the relationship between the maximal 57  rising rate in LV pressure during isovolumetric contraction and EDV during the IVC occlusions. 2.2.5 Statistical analysis  Human data analysis. Summary statistics (mean ± SD) were calculated for each numerical variable. Distribution of data was assessed by the Shapiro-Wilk test. The primary dependent variable was the VAC ratio (continuous, numerical) and the independent variable was the group consisting of SCI-a, SCI-na, and AB (categorical, nominal). The differences between groups were analyzed with analysis of covariance with age as confounding factor. Tukey´s post hoc test was used to assess between-group differences. Categorical variables between the groups were analyzed with a chi-square test.   Animal data analysis. The linear correlation between Ees and VSI was assessed with the Pearson correlation coefficient. To evaluate the agreement between the VSI and invasive Ees, the numerical values were converted into Z-values and calculated two-way mixed-effects consistency intraclass correlation coefficient (ICC). As supplementary analysis, we compared peak blood velocity (PBV), time-to-peak (TTP), VSI and heart rate adjusted VSI (VSI/HR) between sham and SCI, before and after injury by 2-way analysis of variance (group x time). Post hoc pairwise comparison was performed with Sidak’s correction. Additionally, differences in EA, Ees, and EA/Ees (VAC) between sham and SCI were analyzed with unpaired T-tests. All statistical tests were performed at an α level of 0.05. The statistical analyses were performed using SPSS version 22 (SPSS Inc., IBM, Chicago, IL, USA) and Graph Pad v6 (GraphPad Software, CA, USA).  58  2.2.6 Sample size calculation  For the human study, Currie et al. demonstrated systolic function differences in SV, between AB, trained and untrained individuals with tetraplegia. The effect size in SV for the groups was 1.13, which results in a total sample size of 15 with a 90% power. In this study, a total of 44 participants for the analysis was included (11-15 per group). For the animal validation analysis, the sample size calculation was based on the study conducted by Bauer et al.73. They reported a linear correlation of  r=0.82 between invasive Ees and VSI, resulting in an effect size of 0.92 and a total sample size of six animals per group182. Sample size calculations were performed using GPower v3.1.9.2. 2.3 Results  2.3.1 Clinical: anthropometry, blood pressure and physical activity levels.  Age distribution was similar between SCI-na and AB (p=0.563); however, SCI-a were younger compared to AB (p=0.046) and SCI-na (p=0.006). Sex distribution between the groups was similar (p=0.139). Anthropometric characteristics including height, mass, and body surface area (BSA) were not significantly different between the groups. Body mass index (BMI) was lower in SCI-a compared to AB (p=0.002). There was a tendency for SCI-a to present lower BMI compared to SCI-na (p=0.053). BMI between SCI-na and AB was not different (p=0.611). Moderate-to-vigorous LTPA was higher in SCI-a compared to SCI-na (p=0.007), similar between SCI-a and AB (p=0.561) and tended to be higher in AB versus SCI-na (p=0.053) (Table 2.1).  59  Table 2.1 Demographic and physical activity characteristics BMI, body mass index; BSA, body surface area; LTPA; leisure time physical activity. One-way ANOVA with Tukey’s posthoc test for numerical and Chi-square for categorical variables. Unpaired t-test for mild and heavy intensity LTPA. *p<0.05 vs able-bodied, †p<0.05 vs SCI non-athletes.   2.3.2 Hemodynamic, vascular structure and function characteristics.  The three groups showed similar supine SBP, DBP, MAP, pulse pressure (PP) and HR (Table 2.2). TPR was significantly higher in SCI-na compared to AB (p=0.006) and SCI-a (p=0.026), with no difference between AB and SCI-a (p=0.705). However, after age adjustment, TPR was only significantly different between SCI-na and AB (p=0.006). For vascular structure, carotid external diameter (ED) was greater in SCI-na compared to AB (p=0.007) and SCI-a (p=0.013), but BSA and age-adjusted ED was only higher in SCI-na compared to AB (p=0.005). Absolute and BSA-adjusted lumen diameter were similar between groups (p=0.476 and p=0.170, respectively). IMT was higher in SCI-na compared to SCI-a (p=0.051 and AB (p=0.0091) but no longer different after age-adjustment (p=0.977). W:L was increased in SCI-na when compared to SCI-a (p=0.012) and AB (p=0.045), but it was similar between SCI-a and AB (p=0.778). Furthermore, age-adjustment of W:L, although not significant, showed a similar trend being higher in SCI-na compared to SCI-a (p=0.086) and AB (p=0.097). For the vascular function parameters, PSV and SR were greater  Able-bodied  (n=14) SCI Non-Athletes (n=11) SCI Athletes (N=14) p Age, yrs   40.4 ± 13.6 45.0 ± 12.1 30.1 ± 6.3*† 0.007 TSI, yrs         -       19.7 ± 13 12.7 ± 5.1 0.139 Sex (Male), %      71.4 81.8      85.7 0.927 Height, m 1.74 ± 0.08 1.78 ± 0.07   1.8 ± 0.06 0.094 Mass, kg 79.4 ± 12.2 78.4 ± 22.6 67.9 ± 10.9 0.112 BMI, kg/m2 26.1 ± 2.8       24.5 ± 5.8 20.7 ± 2.8* 0.003 BSA, m2 1.94 ± 0.17 1.95 ± 0.26 1.86 ± 0.15 0.489 LTPA   Moderate-to-vigorous, hr·wk   6.4 ± 1.9 1.4 ± 2.2   8.8 ± 2.4† 0.004 60  in SCI-a vs. SCI-na (p<0.001) and AB (p=0.004). Although, after age-adjustment PSV was only different between SCI-a and SCI-na (p=0.027). β, local PWV, EM, DC, and CC were not significantly different between groups (Table 2.2). Table 2.2 Hemodynamic and vascular structure and function between groups.  Able-bodied (n=14) SCI Non-Athletes (n=11) SCI Athletes (N=14) p Age adjusted Hemodynamics    SBP, mmHg 115 ± 12    116 ± 13     110 ± 8  0.282 0.570 DBP, mmHg 66 ± 6      66 ± 9       60 ± 8  0.094 0.587 MAP, mmHg 86 ± 7      86 ± 10       80 ± 7  0.114 0.536 PP, mmHg 49 ± 9      49 ± 11       49 ± 4  0.993 0.905 HR, bpm 60 ± 9      55 ± 10       61 ± 9  0.310 0.579 TPR, mmHgˑminˑL 20 ± 2      28 ± 6* 22 ± 7†  0.004 0.008 Vascular structure    ED, mm   7.3 ± 0.3      8.1 ± 0.7* 7.5 ± 0.4†   0.001 0.006 ED·BSA, mm   6.6 ± 0.4      7.5 ± 0.8*      7.0 ± 0.5   0.006 0.007 Lumen, mm      5.44 ± 0.5    5.63 ± 0.4    5.35 ± 0.4   0.593 0.402 Lumen·BSA, mm   4.9 ± 0.4      5.1 ± 0.5      5.3 ± 0.5   0.091 0.156 IMT, µm       535 ± 89     610 ± 124     524 ± 43†   0.042 0.357 Wall-to-lumen ratio      0.36 ± 0.09  0.45 ± 0.13* 0.33 ± 0.05†   0.011 0.055 Vascular structure    PSV, cm·s        99 ± 19 84 ± 21    124 ±20*† <0.001 0.031 SR, s-1      132 ± 21 103 ± 32*    163 ± 32*† <0.001 0.020 β       5.8 ± 1.9 7.1 ± 2.4      5.8 ± 1.6   0.231 0.510 Local PWV, m·s       5.7 ± 0.7 6.4 ± 1.3      5.5 ± 0.8   0.089 0.779 EM, kpa ˑ 103,    0.417 ± 0.13 .566 ± 0.29  0.426 ± 0.12   0.116 0.402 DC, kpa-1ˑ10-3     33.1 ± 9.7 29.7 ± 12.9    37.0 ± 12.0   0.299 0.998 CC, m2·Pa    1123 ± 375    1022 ± 353   1204 ± 377   0.469 0.795 SBP, systolic blood pressure; DBP, diastolic blood pressure; MBP, mean blood pressure; PP, pulse pressure; HR, heart rate; TPR, total peripheral resistance; ED, external diameter; BSA, body surface area; IMT, intima-media thickness, PSV, peak systolic velocity; SS, shear stress; β, beta stiffness index; PWV, pulse wave velocity; EM, elastic modulus; DC, distensibility coefficient; CC, compliance coefficient. One-way ANOVA with Tukey’s posthoc test. *p<0.05 vs able-bodied, †p<0.05 vs SCI non-athletes.  2.3.2 LV ultrasound derived parameters of structure and function  Left ventricular structure and volume. The absolute LV internal diameter (LVIDd), interventricular (IVST), posterior wall thickness (PWT) in diastole, and relative wall thickness (RWT) did not differ between the groups (p>0.05). After adjustment for BSA, only 61  LVIDd was significantly decreased in SCI-na compared to AB (p=0.032) and indexed LV mass tended to be lower (p=0.094). All cardiac structural parameters were similar between AB and SCI-a. For LV volumes, EDV was lower in SCI-na compared to AB (p=0.004) and similar to SCI-a (p=0.337). No difference was found in ESV between groups. SV was reduced in SCI-na (p=0.001) and SCI-a (p=0.034) compared to AB but no difference was found between SCI-na and SCI-a (p=0.376). Similarly, SVI was reduced in SCI-na compared to AB (p=0.002), but no different between AB and SCI-a (p=0.185). CO was significantly decreased in SCI-na compared to AB (p=0.002), similar between AB and SCI-a (p=0.260). EF did not differ between both SCI groups and AB (p=0.561) (Table 2.3). Pulsed and tissue Doppler. Mitral valve E, A, E/A, and deceleration time were not significantly different between the groups (Table 3.2). Tissue Doppler analysis showed that only Em was decreased in SCI-a (p=0.015) and SCI-na (p=0.033) in comparison to AB. Systolic (S’) and annular late diastolic tissue velocities (A’) did not differ between the groups. The ratio E/Em was significantly higher in the SCI-na compared to AB (p=0.022), but similar between SCI-a and AB (p=0.117). With respect to cardiac cycle times, systolic time (S) was shorter in the SCI-a compared to SCI-na (P=0.039), and no different to AB (p=0.435). IVCT and IVRT showed a tendency to be longer in the SCI-na (p=0.073) (Table 2.3).     62  Table 2.3 Left ventricular structure and volume between able-bodied, athletes and non-athletes with SCI.  Able-bodied (n=14) SCI Non-Athletes (n=11) SCI Athletes (n=13) p Structural LVIDd, mm     44.9 ± 4.4      41.1 ± 5.8        42.2 ± 4.8 0.145 LVIDd · BSA     23.3 ± 1.6 21.1 ± 2.0*        22.8 ± 2.4 0.035 IVST, mm     10.7 ± 1.3 9.5 ± 2.3          9.5 ± 1.5 0.156 IVST · BSA       5.5 ± 0.6 4.8 ± 0.9 5.1 ± 0.6 0.084 PWT, mm     10.1 ± 1.2      10.4 ± 2.3 9.6 ± 1.6 0.475 PWT · BSA 5.2 ± 0.6  5.4 ± 1.3 5.2 ± 0.6 0.800 LV mass, g      161 ± 36       139 ± 58         132 ± 37 0.200 LV mass · BSA, g·m2 83 ± 14 60 ± 24 70 ± 16 0.096 RWT 0.45 ± 0.08 0.50 ± 0.11 0.45 ± 0.08 0.358 Volumetric EDV, mL      118 ± 20   95 ± 10*        106 ± 13 0.004 ESV, mL 48 ± 11         40 ± 7          43 ± 6 0.071 EF, %        59 ± 3      58 ± 3          58 ± 2 0.561 SV, mL 70 ± 10      55 ± 5*          62 ± 9 0.001 SVI, mL        35 ± 3      28 ± 3*          32 ± 6 0.003 CO, L·min 4.1 ± 0.7 3.0 ± 0.6* 3.8 ± 0.6 0.002 Pulsed Doppler E, m·s 0.78 ± 0.16   0.85 ± 0.35   0.8 ± 0.15 0.723 A, m·s 0.49 ± 0.16   0.45 ± 0.07 0.49 ± 0.07 0.572 E/A   1.7 ± 0.47   1.69 ± 0.53 1.64 ± 0.39 0.812 Dec, ms      190 ± 38    202 ± 28        196 ± 68 0.829 Tissue Doppler S’, mm·s 100 ± 18      88 ± 17          92 ± 9 0.155 Em, mm·s 142 ± 25    118 ± 26* 116 ± 17* 0.009 A’, mm·s 100 ± 24   83.3 ± 25 72 ± 15 0.572 E/Em  5.6 ± 0.9     7.3 ± 2.5* 6.8 ± 1.0 0.020 IVCT, ms  66 ± 13      74 ± 9          62 ± 11 0.073 S, ms       296 ± 24    310 ± 35     281.3 ± 25 † 0.049 IVRT, ms  69 ± 10      70 ± 15 59 ± 14 0.093 D, ms  572 ± 141    620 ± 179 609 ± 220 0.770 LVIDd, End-diastolic left ventricular internal diameter; BSA, body surface area; IVST, interventricular septum thickness; PWT, posterior wall thickness; LV, left ventricle; RWT, relative wall thickness; EDV, end-diastolic volume; ESV, end-systolic volume; EF, ejection fraction; SV, stroke volume; SVI, stroke volume index; CO, cardiac output; E, early diastolic filling capacity; A, atrial diastolic filling velocity; Dec, deceleration time; S’, systolic tissue velocity; Em, early diastolic tissue velocity; A’, atrial diastolic tissue velocity; IVCT, isovolumetric contraction time; S, systolic time; IVRT, isovolumetric relaxation time; D, diastolic time. One-way ANOVA with Tukey’s posthoc test. *p<0.05 vs able-bodied, †p<0.05 vs SCI non-athletes.      63  2.3.3 Non-invasive ventricular-arterial coupling  Ees(sb) to assess VAC.  Compared to AB, the pre-ejection period (PEP) tended (p=0.069) to be longer in SCI-na but total systolic time (TSP) was significantly longer in SCI-na compared to SCI-a (p=0.027), and no different to AB (p=0.339). The calculated group-averaged (END(avg)), and normalized LV elastance (END(est)) was similar between groups (p=0.380 and p=0.380, respectively). Likewise, end-systolic blood pressure (ESBP), SV(Doppl), and EA(Doppl) were similar between the groups. Ees(sb) and VAC(sb) showed no statistical difference between the groups (Table 2.4). VSI to assess VAC. In comparison to VAC(sb), determination of VAC using VSI showed significant differences between the groups. Analysis of TTP showed longer times in SCI-na compared to AB (p=0.003) but no different between SCI-a and AB (p=0.125) (Figure 3.1a). The PBV in SCI-na was significantly reduced (p=0.008) in comparison to AB, while it was similar between SCI-a and AB (p=0.386). VSI was significantly decreased in SCI-a (p<0.001) and SCI-na (p=0.043) compared to AB; whereas, VSI tended to be higher in SCI-a (p=0.074) in comparison to SCI-na. HR-adjusted VSI (VSI/HR) showed the same pattern in SCI-na compared to the AB (p<0.05). However, VSI/HR was not significantly different between AB and SCI-a (Table 2.4). When compared to AB and SCI-a, SCI-na presented the highest VAC(VSI) (p<0.001). In addition, VAC(VSI) was significantly lower in SCI-a compared to SCI-na (p=0.017) but tended to be greater than AB (p=0.069).     64  Table 2.4 Non-invasive ventricular-arterial coupling calculated by Ees(sb) and VSI.  Able-bodied (n=14) SCI Non-Athletes (n=13) SCI Athletes (N=13) p Single-beat End-Systolic Elastance PEP, ms    69.9 ± 11       82.3 ± 17.4     71.1 ± 11.5 0.069 TSP, ms  378.5 ± 22.7     396.5 ± 40.0   362.8 ± 27.7 0.035 TND  0.184 ± 0.02     0.209 ± 0.04   0.197 ± 0.03 0.315 END(est)  0.305 ± 0.02     0.209 ± 0.04   0.197 ± 0.03 0.544 END(avg)  0.260 ± 0.05     0.294 ± 0.07   0.272 ± 0.06 0.380 ESBP, mmHg  104.2 ± 10.9     104.8 ± 12.4     98.9 ± 7.3 0.282 SV(Doppler), mL 70.1 ± 16.6 61.4 ± 14.2 66.6 ± 12.5 0.358 Ea(Doppl), mmHg·mL 1.56 ± 0.44 1.76 ± 0.32     1.54 ± 0.31 0.287 Ees(sb) 1.64 ± 0.24 1.83 ± 0.47     1.91 ± 1.1 0.625 VAC(sb) 0.97 ± 0.42 1.00 ± 0.22     0.96 ± 0.21 0.962 Ventricular Stiffness Index TTP, ms   73.3 ± 13.4 92.8 ± 11.0*     83.5 ± 15   0.004 PBV, cmˑs 1038.1 ± 137.3 852.7 ± 107.0*   963.8 ± 170   0.011 Ea(Simp), mmHˑgmL   1.48 ± 0.17 1.9 ± 0.23*  1.61 ± 0.25† <0.001 VSI, cmˑs2     14.7 ± 3.2 9.4 ± 1.9*     12.0 ± 2.9* <0.001 VSI/HR   0.24 ± 0.05 0.17 ± 0.04*  0.19 ± 0.04*   0.002 VAC(VSI)   1.05 ± 0.22 2.1 ± 0.54*  1.44 ± 0.51† <0.001 PEP, pre-ejection period; TSP, total systolic period; TND, ratio of pre-ejection period to total systolic period; END(est), noninvasive estimated normalized  left ventricular elastance at the onset of ejection; END(avg), group-averaged normalized left ventricular elastance at the onset of ejection ; ESBP, end-systolic blood pressure, EA(Doppl), Doppler-derived arterial elastance; Ees(sb), single-beat end-systolic elastance, VAC(sb), single-beat ventricular arterial coupling; TP, time-to-peak velocity; PBV, peak blood velocity; Ea(Simp), Simpson’s derived arterial elastance; VSI, ventricular stiffness index, VAC(VSI), ventricular stiffness index estimated ventricular arterial coupling. One-way ANOVA with Tukey’s posthoc test. *p<0.05 vs able-bodied, †p<0.05 vs SCI non-athletes.  2.3.4 Invasive LV contractility indices in rodents and VSI  Invasive indices of systolic function. One rat in each group was excluded from the final analysis due to a non-optimal Doppler trace.  Ten Wistar rats were included in the analysis. In comparison with sham, T3 SCI animals showed decreased SW, SV, CO and maximal systolic pressure (Pmax) (p<0.05) but no difference in EF (p=0.341) (Table 2.4). Similarly, there were no differences in EA between SCI animals and sham. Regarding load-independent indices of LV contractile function, Ees and +dP/dt max-EDV were significantly reduced in 65  SCI compared to sham animals (p<0.05) but PRSW showed no difference between groups (p=0.325) (Table 3.4). VSI as contractile function index in rats. Group mean values for all indices are provided in Table 2.6. There was a simple main effect for PBV between pre- versus post-injury (p=0.032) (Figure 2.1a). There was an interaction effect between group and baseline-termination on TTP (p=0.023), with post hoc analysis revealing an increase in TTP in SCI animals at termination (p=0.014) (Figure 2.1b). For VSI, a significant interaction effect also occurred between group and time (p=0.008), with a lower VSI in the SCI group at termination (p=0.0116) (Figure 2.1c).  Table 2.5 Catheter derived parameter of systolic function and ventricular-arterial coupling between sham and SCI rats.             Sham (n=5) T3 SCI (n=5)    p SW, mmHgˑmL      16 ± 3          10 ± 3 0.013 SV, µL    131 ± 26          83 ± 17 0.009 CO, mLˑmin   61.5 ± 32.9       37.6 ± 10.0 0.001 Pmax, mmHg 113.5 ± 7.0       97.6 ± 2.6 0.001 EF, %      70 ± 9          64 ± 10 0.341 Ea, mmHgˑmL   0.52 ± 0.04       0.63 ± 0.21 0.321 Ees, mmHgˑmL     2.5 ± 0.51       1.23 ± 0.60 0.011 Ea/Ees   0.21 ± 0.05       0.65 ± 0.40 0.045 PRSW, mmHgˑmL    157 ± 51        123 ± 39 0.325 +dP/dtmax-EDV, mmHgˑsec µL      21 ± 9            9 ± 3 0.031 SW, stroke work; SV, stroke volume; CO, cardiac output; Pes, end-systolic pressure; EF, ejection fraction; Ea, effective arterial elastance; Ees, end-systolic elastance; PRSW, preload recruitable stroke work; +dp/dt max, maximal pressure change rate, EDV; end-diastolic volume.    66  B as elineT ermina tion6 0 07 0 08 0 09 0 01 0 0 0PBV (cm/s)S h amS C I†B as elineT ermina tion1 52 02 53 03 5TTP (ms)*B as elineT ermina tion02 04 06 0VSI (cm/s2) *acbVSI / HRB as elineT ermina tion0 .0 00 .0 50 .1 00 .1 50 .2 0*d Figure 2.1 Peak blood velocity (PBV), time-to-peak (TTP) and ventricular stiffness index (VSI) at baseline and termination in sham (blue) and spinal cord injury (SCI) (green) animals. *Significant post-hoc comparison at that time point following an interaction effect (p<0.05); † significant main effect for time (p<0.05).         67    Table 2.6 Baseline and termination pulmonary artery Doppler parameters.          Sham (n=5)  T3 SCI (n=5)  Baseline Termination Baseline Termination PBV, mmˑs  891.2 ± 72.1   865.6 ± 34.5   898.3 ± 58.7 813.0 ± 91.3† TTP, ms      23.1 ± 3.5     22.2 ± 5.7     20.5 ± 4.6 28.3 ± 6.3† VSI      39.9 ± 5.5     40.5 ± 8.3 46.6 ± 11.6   30.4 ± 4.3†* VSI/HR    0.11 ± 0.02 0.11 ± 0.01 0.13 ± 0.03   0.09 ± 0.01† PBV, peak blood velocity; TTP, time-to-peak; VSI, ventricular stiffness index; HR, heart rate. 2-way ANOVA with Tukey posthoc comparison. *p<0.05 vs sham termination, †P<0.05 vs T3 SCI baseline.  Relationship between VSI and invasive contractile indices. There was a moderate positive correlation between dP/dtmax -EDV and Ees (p=0.053), and a high positive correlation between VSI and Ees (p=0.005). No correlation was found between Ees and PRSW (p=0.38) (Figure 2.1 a-c). ICC analysis showed good agreement between Ees and VSI (ICC 0.880; 95% CI 0.519 to 0.970; p=0.002) and VSI/HR (ICC 0.792, 95% CI 0.164 to 0.948; p=0.014).       68   E S P V RVSI (cm/s2)0 1 2 3 401 02 03 04 05 0 r= 0 .8 0p < 0 .0 1P R S WVSI (cm/s2)0 1 0 0 2 0 0 3 0 001 02 03 04 05 0 r= 0 .3 0p = 0 .3 8d P /d t-E D VVSI (cm/s2)0 1 0 2 0 3 0 4 001 02 03 04 05 0S h a mS C Ir= 0 .6 2p = 0 .0 5abc Figure 2.2 Correlation between maximal rate pressure change-EDV (dP/dt max-EDV) and preload recruitable stroke work (PRSW) and ventricular stiffness index (VSI). Closed blue and green circles represent sham and SCI animals, respectively. Significant relationship p<0.05.  69  2.4 Discussion  The present study evaluated VAC in able-bodied individuals and individuals with chronic SCI who were either non-physically active or wheelchair rugby athletes. The aim of this chapter was to estimate VAC in athletes and non-athletes with SCI with two validated load-independent parameters of cardiac contractility, the single-beat end-systolic elastance Ees(sb)70 and VSI73. Additionally, we retrospectively validated VSI in rats with SCI. The main findings were that VAC estimated with VSI showed a “non-optimal” VAC in the non-athlete SCI group, while the SCI-athletes presented an improved VAC due to greater contractility and a lower EA compared to SCI-na..VAC calculated with Ees(sb) and EA(Doppl) did not show differences between the groups. In rodents, we confirmed that SCI induces systolic dysfunction and ventricular-vascular uncoupling. Furthermore, we found VSI was highly correlated with invasive Ees and moderately with dP/dtmax-EDV suggesting it is an appropriate non-invasive measure of cardiac contractile function in SCI. 2.4.1 Clinical: demographics and blood pressure  Anthropometry. In the present study, BMI differed between SCI groups with SCI-a presenting a lower BMI compared to AB and SCI-na. BMI is commonly used to detect the presence of overweight and obesity183. According to the World Health Organization (WHO) classification, both of the SCI groups fell within normal weight categories (18.5-24.9 kg/m2), with the AB group being categorized as pre-obesity (25.0-29.9 kg/m2)183. A retrospective study conducted in the US reported that SCI presents a similar prevalence of overweight and obesity to that of the general population184. Although both SCI groups showed similar BMI but lower than AB, it has been reported that in SCI there is a lower lean tissue mass and overall body fat percentage185. Moreover, the 30 kg/m2 WHO cut-off could miss to identify 70  obesity in SCI in up to 73.4% of cases186, for that reason, lower BMI cut-off points have been suggested for SCI based on percentage of fat mass and C-reactive protein (inflammation biomarker). Laughton et al.186  proposed a 25 kg/m2 cuff-off value for obesity in SCI, which provides a 90% sensitivity to detect obesity in this population. Using a 25 kg/m2 threshold, we detected a greater prevalence of obesity in SCI-na compared to SCI-a (34% vs 4%, respectively). The BMI group differences observed in this study are in line with those reported by Currie et al., where trained tetraplegics had on average lower mass than untrained tetraplegic and AB. These differences may be explained by the greater levels of physical activity (>15 h/week), greater preservation of muscle mass and reduced gain of body fat in physically active individuals with SCI187. Blood pressure. The supine BP showed no difference between both SCI groups and AB. It is commonly known that individuals with high-level SCI present with low resting BP;  this is more evident in the seated position due to blood pooling in lower limbs and absence of sympathetic vasoconstrictor response below the level of injury92. Indeed, previous studies examining BP in tetraplegia have reported no differences in the supine position111, 188, 189. This similarity of BP between groups can be explained by the lack of a gravitational column in the supine position which elicits a blood redistribution from lower limbs, abdomen and pelvic region, which increase blood volume return to the heart; thus, increasing CO and BP. In the present study, brachial BP measurements were performed after a resting period which allowed for hemodynamic stabilization. Since the main objective of the study was to evaluate resting VAC and given that cardiac ultrasound required for the participants to be in lateral decubitus, no seated BP assessment was performed.  71  2.4.2 Vascular structure and function   Vascular structure. A greater common carotid artery (CCA) diastolic diameter in supine position was found in SCI-na compared to AB, and no difference between SCI-a and AB. Wecht et al.190 reported similar results when they studied the diameter and flow response after a head-up tilt test in tetraplegic, paraplegics, and AB. Specifically that study reported that compared to paraplegics, tetraplegics presented greater CCA diameter and flow than paraplegics; and that both flow and diameter significantly decreased in tetraplegics after a 45° tilt. A possible explanation to the greater CCA diameter in tetraplegia could be a greater elasticity of conduit arteries; however, local arterial stiffness assessed in this study showed no differences versus AB (Further discussed in detail). Another possible explanation is the up-regulation of inducible nitric oxide synthase (iNOS); hence, greater levels of vascular NO. A rodent study conducted by Sangha et al.191 created microgravity conditions – similar to bed rest – where they unloaded the vasculature by elevation of the forelimbs. They reported greater expression of iNOS in the femoral and carotid vascular wall in hindlimb unweighted rats compared to control, which lead to greater vascular NO levels and enhanced vasodilatory response in the upper body vasculature – including the carotid artery. This up-regulation of NO in immobilization studies may explain the increased CCA diameter observed in the SCI-na. There are studies that have reported increased carotid IMT after SCI128, 131, 132, 192, 193; however, their SCI groups consisted of a combination of tetraplegics, paraplegics, and individuals with Spina Bifida with different levels and completeness of injury. Contrarily, Bell et al.194 and Jae et al.195 reported similar IMT values between SCI – composed of paraplegics and tetraplegics – and AB. This heterogeneous combination of individuals in 72  prior studies makes difficult to establish how IMT thickness changes in tetraplegics only.  It is possible that  greater inflammation levels may underlie this response196. In the present study, SCI-a presented similar values to those in AB, and SCI-na exhibited greater IMT when compared to AB. Two major factors that could explain these findings are age and carotid shear rate (SR). SCI-na was older and had the lowest SR values (inversely associated with IMT)197. A slower blood flow transition through the carotid wall could increase transportation of atherogenic particles198, lower endothelial stimulation for eNOS release199, and tissue plasminogen activator (tPA)200– which have anti-atherogenic properties. W:L is a parameter that represents the arterial wall thickness relative to its lumen. In our study, SCI-na showed the greatest W:L compared to AB an SCI-a, while AB and SCI-a showed similar W:L. These results in SCI-a agree with those found by Rowley et al.128 who reported that physically active paraplegics exhibited a decreased carotid W:L compared to AB and sedentary wheelchair SCI. Additionally, they observed in non-active SCI a greater carotid diameter (although not significant), greater IMT and similar W:L compared to AB. The authors attribute the lower W:L in active paraplegics to the beneficial effects of aerobic exercise, which is similar to our findings in that our SCI-a individuals had by far the highest level of weekly MVPA. However, a direct comparison cannot be made between their study and ours due to the difference in the level of the injury. The reasoning for the increased W:L in SCI-na is unclear but could perhaps be explained by a greater wall thickening in SCI secondary to increased inflammation and reduced physical activity.  Vascular function. PSV and SR were higher in SCI-a than SCI-na and AB, with the lowest PSV and SR observed in the SCI-na. Carotid PSV measures the maximal blood flow velocity through the carotid artery during systole. On the other hand, SR is the rate at which two 73  adjacent layers move in respect to each other and assesses the blood flow friction on the arterial wall. Both PSV and SR are positively correlated to contractile function and negatively correlated with blood viscosity and blood vessel diameter181.  Boot et al.201 showed no differences in carotid SR between paraplegics and AB controls; however, their SCI groups consisted of individuals with injuries below T4, where there is an intact cardiac sympathetic control27. The present study included only high-level SCI, which showed that despite a greater carotid diameter, there was a significantly lower PSV and SR that indirectly reflects a reduced contractile function202 and possibly greater blood viscosity due to physical inactivity203.  In the case of SCI-a, the increased levels of PSV and SR are likely a consequence of greater physical activity (increased regional blood flow)203 and a greater contractile function, possibly due to a greater sparing of descending sympathetic input to the CV system204. For the parameters of local stiffness, no difference was found between the groups; although, it has been previously reported that aortic stiffness is increased in SCI when measured by carotid-femoral pulse wave velocity (cfPWV)134, 135, suggesting there is a discrepancy between measurements of local carotid versus regional aortic stiffness. A prior study that evaluated carotid stiffness by DC and β stiffness index in SCI (paraplegic and tetraplegics) also reported no differences as compared to AB205. Given the dependency on upper body musculature for daily life activities after SCI, these findings with respect to no arterial stiffening are perhaps not surprising. 2.4.3 LV structure and function  Prior studies that have investigated LV structure and function post-SCI typically report LV atrophy determined by reduced estimated LV mass, decreased EDV and end-diastolic diameter108. Concerning the LV systolic function, it has been reported that SCI 74  causes a reduced SV and CO, but preserved EF108. In this study, we replicated these findings in the SCI-na group, which showed a reduced indexed LV mass, LVIDd, and IVST. In addition, SV, SVI, and CO were also reduced in SCI-na when compared to AB. Interestingly, although not significantly different vs. either SCI-na or AB, the SCI-a showed a lower reduction in LVIDd, LV mass, EDV, SV, and CO compared to SCI-na. This partial preservation of cardiac structure and function might be explained by the effect of exercise or a partially preserved sympathetic trophic stimuli107. A study by Maggioni et al. reported in SCI endurance athletes that performed 1.5 h/week of aerobic exercise for 5 years a 48% greater LV mass than sedentary lesion-matched SCI individuals206. In another study, West and Krassioukov204 evaluated the preservation of spinal autonomic pathways by sympathetic skin response in Paralympic athletes. They reported that those with partial autonomic SSR>1 had a blood pressure control similar to uninjured individuals. In an exercise intervention study in rats with SCI, intrinsic LV contractile function showed no improvement after passive hindlimb exercise or swimming (upper body). However, West et al.110 found in rats with SCI, that passive hindlimb exercise enhanced cardiac volumes likely via increased blood volume. Taking all this into account, SCI-a may either have preserved their LV function via the effects of prolonged endurance training which would be expected to increase blood volume and preserve LV volumes, or it is possible that these individuals had some sparing in the sympathetic fibers which may enable them to exhibit greater excitatory sympathetic control over the heart and vasculature   For diastolic function, human ultrasound studies have reported a preserved E/A115, 119, 206 and an increased E/Em compared to AB, but within normal ranges119. In the present study, these findings were replicated, as no differences were found in E/A but E/Em was 75  significantly higher in SCI-na versus AB. E/Em is used to estimate LV end-diastolic pressure207. Sharif et al. recently demonstrated that after a rapid saline infusion (i.e. increased preload) no changes were observed in E/Em in SCI individuals, which suggests a compliant LV during diastole122. Further, tissue Doppler analysis in this study revealed similar IVRT compared to AB, which could also suggest a preserved diastolic function. Animal studies that have evaluated diastolic dysfunction through LV catheterization show contradictory results. Squair et al.109 reported no differences in diastolic function assessed by the maximal slope of LV diastolic pressure decrease (dP/dtmin) and time constant of isovolumetric relaxation (tau) after 5 weeks of T2 spinal contusion. On the other hand, Poormasjedi-Meibod et al.170 did find an increased dP/dtmin and tau after 12 weeks of T2 spinal transection, suggesting that time after injury may be key in the development of diastolic dysfunction in SCI. It is important to highlight, however, that dP/dtmin208 and tau209 are load-dependent and the load-independent end-diastolic pressure-volume relationship (EDPVR) was not different from uninjured animals in both studies. On the other hand, tissue analysis in rodents with SCI that have assessed myocardial fibrosis tend to report an increased collagen deposition in the LV 5-6 weeks after injury110, 210, 211. The discrepancy of parameters of diastolic function warrant more research to establish the presence of diastolic impairment after SCI. 2.4.4 Non-invasive methods to assess VAC 2.4.3 VAC in humans after SCI  In this study, we found an uncoupled VAC in SCI-na compared to AB, while SCI-a showed an improved (lower) VAC due to a greater estimated LV contractility and lower EA. We also observed differences between the ability of Ees(sb) and VSI to detect the presence of between-group differences in cardiac contractile function, wherein Ees(sb), which is generally 76  considered the gold-standard for non-invasive measures, showed no difference. Additionally, EA calculated with Simpson’s biplane method showed elevated values in SCI-na compared to AB and SCI-a, whereas EA was similar between SCI-a and AB. An optimal stroke work and ventricular metabolic efficiency are achieved at a VAC ratio of 1.0212, whereas both decline at higher or lower ratios.  With a higher VAC there is a greater BP lability213 (i.e. sensitivity to volume and vascular loading), unfavorable LV energetic efficiency81, decreased cardiac reserve during exercise63, and increased risk of heart failure hospitalization in patients with coronary artery disease214.  It is well-documented that LV contractile function is impaired after SCI in animal studies109, 110, 169. In our human study, the gold-standard Ees(sb) did not show a difference between AB and both SCI groups. This lack of difference could be due to several reasons. Ees is a load-independent parameter of myocardial contractility which is influenced by chamber geometry and passive myocardial stiffening66,69. Borlaug et al.66, reported similar increased Ees(sb) in hypertensive individuals with or without HFpEF, even though contractility and myocardial shortening was reduced in HFpEF. Likewise, they reported increased Ees(sb) in those with structural remodeling compared to those without. In the present study, although not significantly different to AB, the RWT and E/Em were increased in SCI-na suggesting greater diastolic filling pressure possibly due to greater ventricular stiffness, as previously reported by Matos-Souza et al.119. There are several additional reasons that could explain the similarity in Ees(sb) between the groups. First, the BP measurements used in the calculation (SBP, DBP and ESBP) are measured in a supine position, which in SCI have been shown to be similar to AB due to hemodynamic stabilization. Second, SV calculated by Doppler did not reflect previously reported reductions using standard Simpson´s method. 77  Lastly, EF was not different between groups in our study, as in line with previous reports. All these factors could intervene and falsely reflect normal or increased Ees(sb) observed in the SCI individuals of this study.  As an alternative approach to Ees(sb), VSI was used to assess LV contractility and VAC. We observed decreased VSI in SCI-na and SCI-a, but at a lesser extent in SCI-a compared to AB. Although the use of VSI to assess VAC has been reported in only one study in humans68, the measurement of PBV and TTP to calculate VSI have been previously studied. In 1966 Noble et al.215 studied maximal blood acceleration (MVA)  in the LV with an electromagnetic flow meter under different inotropic conditions. They increased contractility by drug infusion and decreased contractility by coronary occlusion. They observed that MVA showed the largest increase after inotropic stimulation and the greatest reduction after coronary occlusion compared to SV, peak flow and dP/dtmax. Subsequently, Bennet et al.216 used continuous-wave Doppler in humans to measure the changes of mean blood velocity and MVA in the ascending aorta whilst changing preload and contractility. They observed that mean aortic velocity was relatively insensitive to preload manipulations and observed a greater increase in MVA compared to SV. Furthermore, Wallmeyer et al.74,  reported a high correlation between PBV and the ratio of PBV/TTP with conventional invasive indices of systolic function. In summary, our study showed that by measuring PBV and TTP (i.e VSI) the presence of contractile dysfunction could be detected in individuals with SCI. The reduction in estimated LV contractility in SCI-na (as assessed with VSI) could be explained by factors such as sympathetic decentralization, chronic unloading (i.e. preload reduction) and upregulation of proteolytic pathways that ultimately lead to atrophic 78  remodeling (reduction in cardiomyocyte length and width)109, 116. A study by Zaglia et al.107 demonstrated that cardiac sympathetic innervation is key to regulate cardiomyocyte size through the cardiomyocyte β2-adrenoceptor activity. Furthermore, to illustrate the impact of LV unloading, the Dallas bed rest study reported that as quickly as 20 days of bed rest can significantly reduce systolic function, in terms of SV and CO, at rest and during exercise in healthy AB individuals105. In animals, similar findings have been reported. A canine study observed a 26% reduction in LV mass after reducing preload by IVC constriction for 10 days217. In SCI, chronic unloading to the heart occurs by lower body blood pooling (splanchnic and lower limbs) due to absence of venoconstriction92, and blood volume depletion secondary to skeletal muscle atrophy218.  For the increased VSI in SCI-a compared to SCI-na, a possible cause may be a greater descending sympathetic sparing. It has been reported that there is a discordance between the motor and sensory completeness of the injury and the integrity of autonomic sympathetic pathways in the spinal cord of SCI athletes204. This was observed in athletes with SCI that were classified as having an incomplete (i.e AIS B) cervical injury, who presented greater preservation of autonomic control as assessed by sympathetic skin response (SSR). The individuals with a positive SSR had greater hemodynamic stability after postural changes, suggesting a greater sympathetic control of the vasculature. To further support this autonomic sparing hypothesis, a study by DeVeau et al.169 in rodents found no improvement in cardiac contractile function after upper-limb and lower-limb 25-day exercise intervention in animals with SCI. Likewise, Currie et al.111, studied the global systolic function and LV mechanics in sedentary (<5 hr of physical activity/week) and physically active (>15 hrs of physical activity/week) individuals with tetraplegia and AB. They observed that both SCI 79  groups showed decreased global systolic function indices and they did not find enhanced systolic function or LV mechanics in sedentary versus trained SCI individuals.  2.4.4 Non-invasive estimation of arterial elastance in humans  This is the first clinical study to assess EA in humans with SCI and the effect of regular moderate-intense physical activity on this parameter. We showed in this study that SCI-na had greater EA, while SCI-a exhibited a lower EA than SCI-na, although still higher than AB. We also demonstrated that the Simpson’s derived EA estimation might be more suitable in the SCI population versus Doppler since the latter was unable to detect differences between groups. This is in line with that reported by Williams et al.108 where they found that non-Simpson’s measurements   were not able to detect  SV differences between SCI and AB. The calculation of SV by Doppler requires measurement of aortic annulus diameter and aortic velocity time integral, which in case of SCI the aortic annulus has been reported to be increased219; thus, possibly overestimating SV in SCI. Studies in animals with SCI have also reported increase EA measured by LV catheterization109, 116, 169. Besides EA, afterload can be assessed by TPR and MAP. Even though MAP was comparable between groups, EA and TPR were elevated in SCI-na and SCI-a. MAP is determined by ventricular (CO) and vascular (TPR) parameters, while EA could better characterize the vascular properties because it integrates arterial peripheral resistance and vascular compliance and impedance. Moreover, EA as afterload index has been validated in animals79 and humans78 and its absolute value is able to predict cardiac functional classification63. In the case of SCI, EA could be increased due to a higher aortic stiffness134, 135, aldosterone-induced arterial remodeling220 and greater resistance to flow as demonstrated in lower body vasculature221. In addition, the positive effect exercise on improving arterial function has been mainly reported with lower body 80  exercise with limited evidence about upper body exercise. In SCI, arm crank ergometry does not elicit blood flow changes to lower body vasculature necessary to improve systemic vascular function143. However, one cross-sectional study reported that SCI hand-cyclists exhibited decreased arterial stiffness compared to age-matched AB222. In this case, the beneficial effect on arterial compliance  may be explained by the improvement of CV risk factors including fasting insulin164, insulin resistance223 and low-grade chronic inflammation161. 2.4.5 Reduced systolic function assessed with VSI in SCI rats  In SCI rats we observed a reduction in the LV’s pressure generating capacity (Pmax) and volumes (SV and CO) in SCI rats compared to sham. Likewise, we observed an elevation in Ea and reduction in cardiac contractility indices Ees and dP/dtmax-EDV. These findings are in line with those reported in previous experimental models of high-level SCI by Squair et al.109, Poormasjedi-Meibod et al.170 and DeVeau et al.169. These findings reflect the effects of sympathetic decentralization with subsequent upregulation of cardiac protein degradation, which lead to LV atrophy and a reduction in systolic function.  To our knowledge, this is the first study that independently validates a non-invasive cardiac contractility index to assess VAC in SCI. By evaluating the cardiac contractility using VSI in rats, we observed that there is a reduction in contractility based on VSI by a combination of reduced PBV and prolonged TTP. Although VSI was measured in the pulmonary trunk, the right and left ventricle are interdependent. The RV share common myocardial fibers with the LV224 and up to 40% of the RV’s systolic pressure results from the contraction of the LV225. This ventricular interdependence was reflected by the strong correlation between VSI on the RV and Ees on the LV. Moreover, due to the spinal injury the 81  loss of supraspinal sympathetic control following SCI alters the contractility of both ventricles. Previous studies using echo in rats with SCI have so far reported only volumetric indices of cardiac function; where VSI could now be used as a complementary tool to examine how cardiac contractility changes with time post-injury in animal studies. 2.4.6 Agreement between invasive Ees and VSI in rats  While this is not the first study that reports the validity of VSI as a non-invasive method to assess contractile function, it is the first validation (against directly measured LV contractile function) in SCI. The direct calculation of Ees requires LV catheterization while decreasing preload to the heart, and given the nature of this technique, its clinical applicability to perform periodical evaluations is limited. To address this limitation, Bauer et al.73 conducted a study that compared Ees and VSI on the LVOT to assess acute contractility changes in sheep. They found that similar to Ees, VSI did not change significantly after increasing preload (i.e. saline infusion), afterload (i.e. angiotensin infusion), or decreasing both preload and afterload (i.e. nitroprusside infusion). Moreover, after performing an acute coronary artery occlusion, VSI was not significantly different from Ees at detecting negative inotropic changes73. On the other hand, the lower correlation between VSI and dP/dtmax-EDV and the absence of correlation with PRSW could be explained by the discrepancy between Ees, dP/dtmax-EDV and PRSW at detecting LV cardiac inotropic changes. For example, Tao et al.226 studied the reduction of cardiac contractility in rats with septic shock. They reported that PRSW did not present the same sensitivity to detect a decreased inotropic state compared to Ees and dP/dtmax-EDV. Furthermore, Nemoto et al.227 evaluated the change in Ees and dP/dtmax-EDV after positive inotropic (i.e dobutamine) and negative (i.e esmolol) reporting that dP/dtmax-EDV changed the most after dobutamine, but at a lesser extent after esmolol 82  administration compared to Ees. This suggests that PRSW and dP/dtmax-EDV may have some limitations at evaluating cardiac contractility. Contrarily, Ees and VSI are able to detect either a decrease or increase in cardiac contractility as shown in the study by Bauer et al. and ours, which explain the high correlation observed between both indices. The validation of a clinical measurement requires that is reproducible and provides a good agreement with standard methods in order to be used as a surrogate228. In the present study, the correlation between Ees and VSI were evaluated linearly by the Pearson’s correlation coefficient, and the agreement with consistency 2-way random effects ICC using z-values of Ees and VSI. Pearson’s coefficient evaluates the strength of correlation while the standard method Bland-Altman analysis the agreement between two measurements229; however, in the latter both parameters must express the same units of measurement. ICC on the other hand, might be more appropriate since it evaluates both the correlation and the agreement between two measurements. In our study, Pearson’s coefficient showed a high positive correlation and ICC a good reliability between VSI and Ees, providing evidence of its validity as cardiac contractile index.           83  Chapter 3. Conclusion  In the present thesis I review the structural and functional changes of the heart and the vasculature after SCI, and the changes that occur during and in response to prolonged exercise. Furthermore, I assessed the cardiac-arterial interaction and mechanical efficiency by VAC in sedentary and elite wheelchair rugby athletes compared able-bodied. We found that SCI has a detrimental effect on VAC caused by reduced contractility and increased resistance to blood flow. Our findings in the SCI-athletic group imply that VAC may be improved by performing regular moderate-intense exercise which reduces resistance to flow and hence, cardiac function. We further validated our non-invasive findings in humans with load-independent contractility indices in rats with SCI. 3.1 Implications and future directions  The evaluation of the interaction between the heart and the vasculature is useful to better understand pathophysiological mechanisms of heart failure, exercise capacity, and risk of CVD after SCI. Physical activity may provide a therapeutic tool to improve the interaction between the heart and the vascular system. This is key in SCI individuals to improve their overall cardiac function. Future directions from this study include conducting an exercise intervention to assess VAC changes in SCI individuals, as well as reducing EA as a target to improve VAC. To further validate VSI and VAC(VSI), a prospective study would be necessary to establish their capability to predict CV outcomes. 3.2 Study strengths and limitations  In this study, we provide a non-invasive approach to assess VAC in humans that could help track therapeutic interventions aiming to improve CV function after SCI. We 84  achieved this by testing a large sample size of cervical AIS A-B SCI individuals, the inclusion of highly trained SCI athletes and the use of a relatively novel index of cardiac contractility. Moreover, our human findings were validated with gold-standard LV contractile measurements in an SCI animal model. The limitations of this study include the age difference between the groups which was statistically adjusted. Furthermore, the integrity of autonomic pathways was not assessed which could support the hypothesis of greater sympathetic preservation in SCI-a. For the limitation of VSI, the presence of valvulopathies or any type of LVOT obstruction could falsely augment PBV and VSI. Moreover, one of the limiting factors of using EA as afterload index is that it does not account for the arterial reflected waves that can alter systolic loading sequence, which aggravates LV dysfunction.          85  Bibliography  1. Weinhaus AJ, Roberts KP. Anatomy of the human heart.  Handbook of cardiac anatomy, physiology, and devices: Springer; 2009. p. 59-85. 2. Ross MH, Pawlina W. Histology: Lippincott Williams & Wilkins; 2006. 3. Mescher AL. Junqueira's basic histology: text and atlas: Mcgraw-hill; 2013. 4. Ovalle WK, Nahirney PC. Netter's Essential Histology E-Book: Elsevier Health Sciences; 2013. 5. Filipoiu FM. The Structure of the Heart.  Atlas of Heart Anatomy and Development. 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