The development of neuromuscular fatigue during exercise in healthy older individuals and patients with heart failure with a preserved ejection fraction

The purpose of this dissertation was to examine the development of neuromuscular fatigue during exercise and the excitability of the corticospinal motor pathway in healthy aging and in patients with heart failure and a preserved ejection fraction (HFpEF). In the first study, I examined the relationship between the degree of muscle activation and the excitability of the corticospinal pathway (i.e., the primary motor pathway in humans) during isometric knee extensor and cycling exercise. Briefly, I found that increasing levels of muscle activation progressively facilitates the corticospinal pathway via modulating the excitability of the spinal motoneuron pool and this relationship appears to be independent of contractile modality (i.e., isometric small muscle mass vs. dynamic large muscle mass exercise). Based on these findings, I concluded that it is critical to control for the level of muscle activation when examining corticospinal excitability during or after exercise. The second study sought to determine the influence of aging on the development of neuromuscular fatigue and exercise-induced corticospinal alterations during cycling (characterized by large cardiopulmonary stress) or rhythmic dynamic single-joint knee extension (characterized by minimal cardiopulmonary stress). Following cycling and single-joint exercise, older participants demonstrated an enhanced development of neuromuscular fatigue during exercise at a given absolute intensity. However, compared to their younger counterparts, the old developed less central fatigue during exercise performed at the same relative intensity. Active muscle mass had little influence on the discrepancy in the exercise-induced development of neuromuscular fatigue between young and old individuals, indicating the influence of age on the development of fatigue may be more dependent on exercise intensity (i.e., absolute vs. relative comparisons) rather than exercise involving different degrees of active muscle mass. Additionally, corticospinal excitability was unaltered by exercise in either condition or group, indicating aging does not alter the influence of exercise on corticospinal excitability and, consequently, its role in determining exercise-induced fatigue. The third study aimed to elucidate the impact of HFpEF on corticospinal excitability and the development of neuromuscular fatigue during exercise. During dynamic exercise performed at the same relative intensity, patients with HFpEF only completed approximately one-half of the work performed by healthy control participants, but developed a similar degree of end-exercise neuromuscular fatigue compared to their healthy counterpart. When examined following exercise at the same absolute intensity, HFpEF patients demonstrated a greater degree of both peripheral and central fatigue. The greater fatigue in the HFpEF was not related to an impaired motor pathway as corticospinal excitability was unchanged following exercise in both groups. Based on these findings, I concluded that patients with HFpEF are characterized by a substantially compromised fatigue resistance during physical activity. In summary, this dissertation characterizes the development of neuromuscular fatigue and alterations within the corticospinal pathway during exercise in health and disease.

THE DEVELOPMENT OF NEUROMUSCULAR FATIGUE DURING EXERCISE IN HEALTHY OLDER INDIVIDUALS AND PATIENTS WITH HEART FAILURE WITH A PRESERVED EJECTION FRACTION by Joshua Clewell Weavil A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Kinesiology The University of Utah May 2017 Copyright © Joshua Clewell Weavil 2017 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL STATEMENT OF DISSERTATION APPROVAL The dissertation of Joshua Clewell Weavil . has been approved by the following supervisory committee members: and by Markus Amann , Chair 03-03-2017 . Russell S. Richardson , Member 03-03-2017 . John David Symons , Member 03-03-2017 . Jose Nativi-Nicolau , Member 03-03-2017 . Christopher J. McNeil , Member 03-03-2017 . Date Approved Timothy A. Brusseau, Jr. Department/College/School of Date Approved Date Approved Date Approved Date Approved , Chair/Dean of the Kinesiology and by David B. Kieda, Dean of The Graduate School. ABSTRACT The purpose of this dissertation was to examine the development of neuromuscular fatigue during exercise and the excitability of the corticospinal motor pathway in healthy aging and in patients with heart failure and a preserved ejection fraction (HFpEF). In the first study, I examined the relationship between the degree of muscle activation and the excitability of the corticospinal pathway (i.e., the primary motor pathway in humans) during isometric knee extensor and cycling exercise. Briefly, I found that increasing levels of muscle activation progressively facilitates the corticospinal pathway via modulating the excitability of the spinal motoneuron pool and this relationship appears to be independent of contractile modality (i.e., isometric small muscle mass vs. dynamic large muscle mass exercise). Based on these findings, I concluded that it is critical to control for the level of muscle activation when examining corticospinal excitability during or after exercise. The second study sought to determine the influence of aging on the development of neuromuscular fatigue and exercise-induced corticospinal alterations during cycling (characterized by large cardiopulmonary stress) or rhythmic dynamic single-joint knee extension (characterized by minimal cardiopulmonary stress). Following cycling and single-joint exercise, older participants demonstrated an enhanced development of neuromuscular fatigue during exercise at a given absolute intensity. However, compared to their younger counterparts, the old developed less central fatigue during exercise performed at the same relative intensity. Active muscle mass had little influence on the discrepancy in the exercise-induced development of neuromuscular fatigue between young and old individuals, indicating the influence of age on the development of fatigue may be more dependent on exercise intensity (i.e., absolute vs. relative comparisons) rather than exercise involving different degrees of active muscle mass. Additionally, corticospinal excitability was unaltered by exercise in either condition or group, indicating aging does not alter the influence of exercise on corticospinal excitability and, consequently, its role in determining exercise-induced fatigue. The third study aimed to elucidate the impact of HFpEF on corticospinal excitability and the development of neuromuscular fatigue during exercise. During dynamic exercise performed at the same relative intensity, patients with HFpEF only completed approximately one-half of the work performed by healthy control participants, but developed a similar degree of end-exercise neuromuscular fatigue compared to their healthy counterpart. When examined following exercise at the same absolute intensity, HFpEF patients demonstrated a greater degree of both peripheral and central fatigue. The greater fatigue in the HFpEF was not related to an impaired motor pathway as corticospinal excitability was unchanged following exercise in both groups. Based on these findings, I concluded that patients with HFpEF are characterized by a substantially compromised fatigue resistance during physical activity. In summary, this dissertation characterizes the development of neuromuscular fatigue and alterations within the corticospinal pathway during exercise in health and disease. iv TABLE OF CONTENTS ABSTRACT ....................................................................................................................... iii LIST OF FIGURES .......................................................................................................... vii LIST OF TABLES ........................................................................................................... viii ACKNOWLEDGEMENTS ............................................................................................... ix CHAPTER 1 ....................................................................................................................... 1 1 INTRODUCTION ........................................................................................................ 1 Initiating Voluntary Movement.......................................................................... 2 The Corticospinal Tract...................................................................................... 3 Spinal Motoneurons ........................................................................................... 4 The Neuromuscular Junction and Muscular Contraction................................... 7 Testing the Excitability of the Corticospinal Pathway ....................................... 8 Assessments of Exercise-Induced Fatigue ....................................................... 15 Aim for Study 1 ................................................................................................ 18 Aim for Study 2 ................................................................................................ 20 Aim for Study 3 ................................................................................................ 22 Summary of Specific Aims of Dissertation ..................................................... 23 References ........................................................................................................ 24 2 INTENSITY-DEPENDENT ALTERATIONS IN THE EXCITABILITY OF CORTICAL AND SPINAL PROJECTIONS TO THE KNEE EXTENSORS DURING ISOMETRIC AND LOCOMOTOR EXERCISE ...................................... 40 Abstract ............................................................................................................ 41 Introduction ...................................................................................................... 41 Methods ............................................................................................................ 42 Results .............................................................................................................. 44 Discussion ........................................................................................................ 46 Grants ............................................................................................................... 49 Disclosures ....................................................................................................... 49 Author contributions ........................................................................................ 49 References ........................................................................................................ 49 3 THE IMPACT OF AGE ON THE DEVELOPMENT OF FATIGUE DURING LARGE AND SMALL MUSCLE MASS EXERCISE .............................................. 51 Abstract ............................................................................................................ 52 Introduction ...................................................................................................... 52 Methods ............................................................................................................ 55 Results .............................................................................................................. 63 Discussion ........................................................................................................ 66 References ........................................................................................................ 72 4 HEART FAILURE WITH PRESERVED EJECTION FRACTION IMPAIRS PERIPHERAL HEMODYNAMICS AND RESULTS IN EXAGGERATED NEUROMUSCULAR FATIGUE .............................................................................. 83 Abstract ............................................................................................................ 84 Introduction ...................................................................................................... 85 Methods ............................................................................................................ 87 Results .............................................................................................................. 94 Discussion ........................................................................................................ 98 References ...................................................................................................... 103 5 CONCLUSION ......................................................................................................... 117 vi LIST OF FIGURES Figures 1.1 Testing the excitability of the corticospinal pathway ..................................................39 2.1 Representative raw corticospinal [maximal M-waves (Mmax), motor-evoked potentials (MEP), and cervicomedullary-evoked potentials (CMEP)] traces obtained during each isometric contraction strength and cycling workload ..................................................45 2.2 Electromyographic (EMG) activity and corticomotoneuronal-evoked responses of the vastus lateralis and rectus femoris during isometric knee extensor contractions .......46 2.3 EMG activity and CMEP of the vastus lateralis and rectus femoris during cycling exercise ........................................................................................................................47 2.4 Changes in corticomotoneuronal (area of MEP normalized to Mmax) and αmotoneuronal (CMEP normalized to Mmax) excitability of the vastus lateralis (A) and rectus femoris (B) in response to a given increase in voluntary EMG during cycling and isometric knee extensor exercise ...........................................................................48 3.1 Schematic illustration of the exercise protocols ..........................................................78 3.2 Hemodynamic response to knee extensor exercise in young and old participants ......79 3.3 Central and peripheral fatigue induced by single-leg knee extensor exercise .............80 4.1 Schematic illustration of the exercise protocol ..........................................................109 4.2 Hemodynamic response to rhythmic knee extensor exercise ....................................110 4.3 Central and peripheral fatigue induced by rhythmic knee extensor exercise ............111 4.4 Second-by-second plots of cardiovascular responses during isometric exercise.......112 4.5 Central and peripheral fatigue during intermittent isometric exercise.......................113 LIST OF TABLES Tables 2.1 Raw and normalized Mmax, MEPs, and CMEPs obtained during various isometric contraction strengths of the knee extensors .................................................................43 2.2 Raw and normalized Mmax, MEPs, and CMEPs obtained during various cycling workloads .....................................................................................................................44 3.1 Descriptive characteristics of young and older participants ........................................81 3.2 Cardiopulmonary and metabolic responses during the final minute of rhythmic knee extension ......................................................................................................................82 4.1 Descriptive characteristics of healthy controls and patients with HFpEF .................114 4.2 Medications and echocardiography based characteristics of HFpEF patients ...........115 4.3 Cardiopulmonary and metabolic responses during the final minute of rhythmic knee extension performed at the same relative and absolute intensity...............................116 ACKNOWLEDGEMENTS First and foremost, I would like to thank my wife Taylor, and daughter Lucy, for the love and support they have provided during the pursuit of my doctoral degree. Their ability to put a smile on my face and help me maintain focus on the important things in life allowed me to prevail through the most challenging times. Furthermore, I want to recognize my parents, Chip and Robin Weavil (along with brothers Chase and Cody Weavil), for the unconditional guidance they consistently provide. Through leading by example, my family has made me into the person I am today and I am eternally grateful. I would also like to thank the members of my graduate committee: Drs. McNeil, Nativi, Richardson, and Symons. Each member has contributed an invaluable amount of support and time to the development of my academic career. Additionally, Dr. Amann, my committee chair, has been an integral part of my graduate education by providing endless patience and guidance, for this I am forever indebted. Through the constructive criticism and unique perspective each committee member provided, I have been able to grow both personally and professionally. Finally, I would like to thank the members of the Utah Vascular Research Laboratory and the volunteers who participated throughout my dissertation projects. As John Donne once wrote "no man is an island," and without their generous contributions I would not be where I am today. x CHAPTER 1 INTRODUCTION 2 Fatigue is a psychophysical state which can originate from occupational and recreational activities and results in a decreased physical and/or cognitive performance (Romani, 2008). While both physical and cognitive fatigue are prominent topics of study, the current investigations will focus on the physical manifestation of fatigue, defined as an exercise-induced reduction in the force/power-generating capacity of a muscle or muscle group (Vollestad, 1997). Classically, fatigue has been categorized by both peripheral and central components, which are not mutually exclusive (Thomas et al., 2015). Peripheral fatigue is characterized by reversible impairments at or distal to the neuromuscular junction (Gandevia, 2001), including alterations at the neuromuscular junction (Sieck & Prakash, 1995), neuromuscular propagation (Bellemare & Garzaniti, 1988; Milner-Brown & Miller, 1986), and the excitation-contraction coupling process (Allen, Westerblad, Lee, & Lannergren, 1992). Central fatigue entails various processes within the central nervous system (CNS) that reduce neural drive to the muscle and, subsequently, voluntary movement (Gandevia, 2001; Taylor, Amann, Duchateau, Meeusen, & Rice, 2016). Collectively, any combination of peripheral and/or central fatigue can ultimately compromise physical activity and/or athletic performance. Initiating Voluntary Movement When voluntary movement is desired, high order motor areas of the brain (e.g., parietal/premotor cortices and cerebellum) organize a movement plan which is then relayed to the primary (Brodmann's area 4) and supplementary (Brodmann's area 6) motor cortices (2013), hereafter termed the motor cortex. Cell bodies of the motor cortex integrate and 3 transmit the neural impulse through corticospinal axons and other descending tracts (briefly acknowledged below), which terminate in the ventral horn of the spinal cord onto alpha motoneurons directly, or indirectly via interneurons. If the synaptic input is great enough to cause motoneuronal excitation, the neural impulse then propagates down the axon of the motoneuron, across the neuromuscular junction, and arrives on the target skeletal muscle fibers resulting in muscular contraction. As such, the initiation and execution of voluntary movement can be simplified by identifying the interplay between the three primary sites of action: 1) the corticospinal tract (including the motor cortex and descending axons to the spinal motoneuron), 2) the spinal motoneurons, and 3) the neuromuscular junction leading to muscular contraction. The Corticospinal Tract The corticospinal tract represents the highest order of motor function in humans, consisting of cortical cell bodies located in layer V of the motor cortex (upper motor neurons). The motor cortical cells are somatotopically organized by the innervation of specific muscle groups. Axons of these cell bodies extend down the internal capsule and through the midbrain, pons, and pyramids of the medulla. After decussation of approximately 90% of the fibers at the medulla, corticospinal axons enter the spinal cord and terminate on alpha motoneurons in laminae V-VII of the ventral horn (lower motor neurons). The remaining 10% of axons eventually cross over to the contralateral side via the white commissure of the spinal cord at the level of the alpha motoneuron. Although other extrapyramidal tracts, such as the rubrospinal and reticulospinal tracts, may be 4 involved in movement (Haines, 2013), 60-80% of corticospinal axons originate from the motor cortex, thus constituting the primary pathway for voluntary movement (Haines, 2013). The primary site for modulation in the corticospinal tract is the motor cortical cell, which can influence the transmission of neural impulses from the higher brain centers to the spinal motoneuron. Excitatory and inhibitory interneurons alter cortical plasticity when activated via neurotransmitters, such as γ-aminobutyric acid (GABA, inhibitory; Bowery, 1989), serotonin (facilitatory; Nitsche et al., 2009) and dopamine (inhibitory; Ziemann, Tergau, Bruns, Baudewig, & Paulus, 1997; for review on pharmacological effects see W. Paulus et al., 2008). These neurotransmitters, predominately the GABA subtypes, are believed to play a role in intracortical inhibition (Nakamura, Kitagawa, Kawaguchi, & Tsuji, 1997; Sanger, Garg, & Chen, 2001), intracortical facilitation (Kujirai et al., 1993), and the brief interruption of voluntary electromyographic activity (EMG) following cortical stimulation, termed the silent period (Inghilleri, Berardelli, Cruccu, & Manfredi, 1993; Inghilleri, Berardelli, Marchetti, & Manfredi, 1996). Spinal Motoneurons The spinal motoneuron represents the final common pathway in motor processing (Sherrington, 1906). Three categories of motoneurons exist within the ventral horn of the spinal cord: alpha motoneurons which innervate extrafusal fibers (i.e., skeletal muscle), gamma motoneurons which innervate intrafusal fibers (i.e., within muscle spindles), and less common beta motoneurons which innervate both extra- and intrafusal fibers (Manuel 5 & Zytnicki, 2011). A so-called motor unit consists of an alpha motoneuron and the skeletal muscle fibers innervated. Human motor units are typically classified into three subgroups: Type I (slow contracting; small neuron size), Type IIA (fast contracting, fatigue resistant; intermediate neuron size), and Type IIB (fast contracting, fast fatigue; large neuron size). Specific physiological parameters such as force production, contraction speed, ATP concentration, number of innervated fibers, and myosin light chain isoforms are associated with each motor unit (Burke, Levine, & Zajac, 1971). In contrast to alpha motoneurons, gamma motoneurons innervate intrafusal fibers. Specifically, while group Ia afferents are sensitive to lengthening and/or shortening velocity of a muscle fiber, group Ib afferents exhibit activity during changes in muscle tension. Consequently, gamma motoneurons relay information about the contractile state of a muscle. In addition to the neural input from descending pathways, spinal interneurons and segmental afferents (group I-IV) act to regulate the excitatory state of the alpha motoneuron. By altering the intrinsic properties of the motoneuron (e.g., altering the refractory after-hyperpolarization period), neural input to the motoneuron can lead to a greater, or lesser, likelihood for neuronal discharge (Matthews, 1999). Specifically, the motoneuron response to synaptic input, that is, the excitability of the motoneuron, can be augmented via input from the Ia pathway (G. Macefield, Hagbarth, Gorman, Gandevia, & Burke, 1991; G. Macefield, Gandevia, Bigland-Ritchie, Gorman, & Burke, 1993) augmented (Martin, Gandevia, & Taylor, 2006) or diminished by the input of thin diameter group III-IV muscle afferents (Garland, 1991; Kniffki, Schomburg, & Steffens, 1980) and Renshaw cells (Hultborn, Pierrot-Deseilligny, & Wigstrom, 1979; Loscher, Cresswell, & Thorstensson, 1996b). Group III-IV muscle afferents can also indirectly influence 6 motoneuronal excitability by presynaptically inhibiting Ia facilitation during exercise (Rossi, Decchi, & Ginanneschi, 1999) or by attenuating the hyperpolarization from Renshaw cells (Windhorst, Kirmayer, Soibelman, Misri, & Rose, 1997; Windhorst, MeyerLohmann, Kirmayer, & Zochodne, 1997). Group Ib afferents may have inhibitory or excitatory influences on the motoneuron depending on the motor pool examined during movement (Eccles, Eccles, & Lundberg, 1957). In regards to the more distal aspect of motoneuronal excitability, the repetitive activation of motoneurons can lead to an insufficient release, or depletion, of neurotransmitters (e.g., acetylcholine) from the synaptic vesicles. This has the potential to compromise synaptic efficacy (Otsuka, Endo, & Nonomura, 1962; Redman & Silinsky, 1994; Wu & Saggau, 1997; Zucker & Regehr, 2002) which has been suggested to account for some of the decrease in motoneuronal excitability during and immediately after exercise (Gandevia, Petersen, Butler, & Taylor, 1999; Petersen, Taylor, Butler, & Gandevia, 2003). If the summation of excitatory input exceeds inhibitory input, the alpha motoneuron will depolarize. Once depolarization reaches the threshold of excitation, an action potential is generated. This response is often referred to as an ‘all or none response' in the sense that an action potential is only generated when, and not until, the threshold is reached. While having similar voltage thresholds for excitation (~-55 mV), small motor neurons have a higher input resistance than their larger counterparts (Eccles, Eccles, & Lundberg, 1958). The higher resistance results in a larger voltage drop for a given neural input in small motor neurons (Henneman, Somjen, & Carpenter, 1965). Subsequently, small motoneurons reach the excitation threshold at a lower neural input than larger motoneurons. This characteristic provides the basis for the Henneman size principle, which 7 states that motor units are recruited according to their size, from the smallest to largest. Therefore, smaller, fatigue-resistant motor units are more susceptible to discharge at lower levels of neural input and thus are the first to engage during voluntary activity compared to the larger, more fatigable motor units (Henneman, 1957). Additionally, the reverse is true such that larger motoneurons are the first to derecruit as neural input is reduced (Henneman et al., 1965). Importantly, the size principle holds true even during fatiguing exercise (Adam & De Luca, 2003). The Neuromuscular Junction and Muscular Contraction Alpha motoneurons innervate the skeletal muscle fiber at the neuromuscular junction. Once activated, neural impulses proceed down the axon of the motoneuron and arrive at the presynaptic terminal. Depolarization of the presynaptic terminal causes an influx of calcium and the fusion of neurotransmitter-filled vesicles to the presynaptic membrane (Katz & Miledi, 1965; Miledi, 1973). These vesicles release acetylcholine into the synaptic cleft, which, after attaching to nicotinic acetylcholine receptors, open ion channels on the postsynaptic terminal. The resultant voltage change results in an action potential which is propagated along the sarcolemma and down the transverse tubules (i.e., T-tubules) of the muscle fiber activing voltage-gated dihydropyridine and ryanodine receptors eliciting the release of calcium from the sarcoplasmic reticulum. The calcium released from the sarcoplasmic reticulum (Robin & Allard, 2012) binds to troponin C, causing a conformational change in tropomyosin. The thick myosin filament then binds to the thin actin filament, forming a cross bridge resulting in force production (Fitts, 1994). 8 Testing the Excitability of the Corticospinal Pathway The efficacy of the corticospinal pathway to relay neural signals depends on the excitability of its segments, namely the corticospinal tract, the spinal motoneuron, and the neuromuscular junction/skeletal muscle fiber (Martin, Gandevia, et al., 2006). An impairment occurring at any segment has the potential to diminish neural drive to the muscle and, consequently, muscle activation and voluntary movements (Martin, Gandevia, et al., 2006; Sidhu et al., 2014; Taylor, Petersen, Butler, & Gandevia, 2000; Taylor, Todd, & Gandevia, 2006). Therefore, in order to understand the development of fatigue, it is important to evaluate the effect of exercise on the three segments of the corticospinal pathway. The methodologies used to determine exercise-induced changes at the 1) corticospinal tract, 2) spinal motoneuron, or 3) neuromuscular junction/skeletal muscle fiber will be presented in the following paragraphs. The corticospinal tract: In 1954, Patton and Amassian demonstrated that electrical stimulation of the motor cortex in monkeys produces a sequence of descending volleys (Patton & Amassian, 1954). The first of these volleys, termed the D wave, is the result of direct activation of corticospinal axons and occurs a few milliseconds prior to the first indirect wave, or I wave. The prolonged latency of the I wave is thought to be the result of transsynpatic activation of corticospinal neurons and/or interneurons (for review see Terao & Ugawa, 2002). Five years after the electrical stimulation of the motor cortex in humans (Merton & Morton, 1980), Barker, Jalinous, and Freeston (1985) demonstrated that magnetic stimulation of the motor cortex using transcranial magnetic stimulation (TMS) can be a less painful alternative to electrical cortical stimulation. Unlike electrical cortical stimulation, TMS primarily results in I waves (Rothwell, Thompson, Day, Boyd, & 9 Marsden, 1991). Briefly, TMS (Figure 1.1) utilizes a transient magnetic field which induces an electrical current underneath the coil in nearby neurons (Rothwell et al., 1991). When applied to the motor cortex, a short-latency (~12-30 ms in limb muscles) EMG response termed the motor-evoked potential (MEP) can be observed in the contralateral limb muscle of interest. The area, or amplitude, of the MEP indicates the state of excitability for the whole motor pathway (Day et al., 1989; Rothwell et al., 1991) but alone cannot distinguish between the excitability of each individual segment. Importantly, corticospinal projections to contralateral motoneurons of muscles in both the upper and lower limbs are predominately monosynaptic (Brouwer & Ashby, 1990; Day et al., 1987) and independent of presynaptic inhibition (Nielsen & Petersen, 1994). In addition to the MEP, if delivered during a voluntary contraction, TMS of the motor cortex, at sufficient stimulator intensity, induces a brief (typically <300 ms) pause in voluntary EMG activity, termed the silent period. It has been suggested that the early portion of the silent period (~50 ms) is related to spinal mechanisms while the latter, beyond 50 ms, is attributable to inhibition within the cortex (Chen, Lozano, & Ashby, 1999; Inghilleri et al., 1993). However, it has recently been proposed that the spinal portion of the silent period may account for a longer segment than originally believed (Yacyshyn, Woo, Price, & McNeil, 2016). As fatigue develops, the silent period lengthens in duration (Gandevia, Allen, Butler, & Taylor, 1996; Hilty, 2011; Taylor, Butler, Allen, & Gandevia, 1996) indicating increased spinal and/or intracortical inhibition with fatigue. This prolongation may be partially attributed to the activity of group III/IV muscle afferents as the blockade of these muscle afferents prevents an exercise-induced increase in the silent period (Hilty, 2011). 10 In addition to the silent period, inhibitory circuits within the motor cortex can be examined by use of paired transcranial magnetic stimuli. This technique consists of a conditioning stimulus followed by a test stimulus at various interstimulus intervals and stimulator intensities. The magnitude of the response evoked by the conditioned stimulus is compared to the response elicited by the test (nonconditioned) stimulus to provide a measure of the excitatory or inhibitory processes. Frequently, a subthreshold conditioning stimulus proceeds a suprathreshold stimulation at an interstimulus interval of either 1-4 ms (short-interval intracortical inhibition, SICI) or 6-20 ms (intracortical facilitation, ICF; Di Lazzaro et al., 1998b; Kujirai et al., 1993; Sanger et al., 2001; Ziemann, Rothwell, & Ridding, 1996). Alternatively, if a suprathreshold conditioning stimulus is delivered 50200 ms prior to a test stimuli, long-interval intracortical inhibition (LICI) is observed (Valls-Sole, Pascual-Leone, Wassermann, & Hallett, 1992; Wassermann et al., 1996). Intracortial inhibition, as estimated by the silent period, has been demonstrated to increase (McNeil, Martin, Gandevia, & Taylor, 2009) after fatiguing exercise. However, it has been demonstrated that LICI is highly related to changes at the spinal level and may not represent cortical changes as originally postulated (McNeil, Giesebrecht, Gandevia, & Taylor, 2011; McNeil et al., 2009). Spinal motoneurons: The excitability of motoneurons can be explored through many different methods, such as the Hoffmann reflex (H-reflex), F-wave, V-wave, tendon tap, and direct magnetic or electrical activation of the corticospinal tract axons (for review see McNeil, Butler, Taylor, & Gandevia, 2013). The H-reflex and direct activation of the corticospinal axons represent the most robust method for testing spinal motoneuronal excitability (McNeil et al., 2013). The following paragraphs offer a brief discussion of 11 these techniques. The H-reflex is a spinal reflex produced by the activation of large diameter Ia afferents, via low intensity peripheral nerve stimulation (PNS), resulting in a monosynaptic excitation of alpha motoneurons (Hoffmann, 1918). While a similar reflex is present in the tendon tap reflex, the H-reflex does not involve muscle spindle influences resulting from tissue deformation. With increasing PNS intensity, a recruitment curve can be generated for the H-reflex and, when above motor threshold, the muscle compound action potential (M-wave). As the M-wave is the result of direct axonal stimulation, the M-wave precedes the H-reflex. At high stimulation intensities, up to intensities resulting in a maximal Mwave (Mmax), the H-reflex declines due to the orthodromic afferent response colliding with the antidromic motor response (Knikou, 2008). Comparing the maximal H-reflex response (Hmax) to the Mmax provides an estimate of the motoneuron pool involved in the H-reflex (McNeil et al., 2013). However, it is important to note that while the H-reflex follows the size principle, stimulation intensities evoking a Mmax result in the simultaneous activation of all axons near the stimulation site. Therefore, the same motoneurons are not active during Hmax and Mmax. Although the H-reflex is frequently used as a method to directly test motoneuronal excitability, interpretation of the reflex may be confounded by presynaptic inhibition (Hultborn, Meunier, Pierrot-Deseilligny, & Shindo, 1987), reciprocal inhibition (Hoffmann, 1952), and post-activation depression (Crone & Nielsen, 1989) (for review see Zehr, 2002). Another method of testing motoneuronal excitability is through the noninvasive direct stimulation of the corticospinal axons (Ugawa, Rothwell, Day, Thompson, & Marsden, 1991). This stimulation can be either magnetic or electrical (Taylor, 2006) and 12 evokes a short-latency cervicomedullary motor evoked potential (~8-20 ms in the limbs, CMEP) or a thoracic motor evoked potential (TMEP), depending on the site of stimulation. Specifically, cervicomedullary stimulation (CMS) consists of a high-voltage stimulation applied to either side of the mastoid process at the level of the cervicomedullary junction (i.e., medullary decussation; Taylor, 2006), whereas thoracic stimulation is delivered with the cathode between T3 and T4 of the spine and the anode 5-10 cm above the cathode (Martin, Butler, Gandevia, & Taylor, 2008; Ugawa, Genba-Shimizu, & Kanazawa, 1995). It has been noted that thoracic stimulation provides a valuable alternative for stimulating the corticospinal tract innervating the lower limbs, as CMS may not be sufficient to evoke EMG potentials in the lower limbs in all individuals (Ugawa et al., 1991). However, as there are only limited data currently available examining thoracic stimulation, electrical CMS will be the primary method for investigating motoneuronal excitability for this dissertation. CMS produces only one descending volley which can be diminished, and sometimes abolished, via antidromic collision from PNS (Berardelli, Inghilleri, Rothwell, Cruccu, & Manfredi, 1991). Additionally, CMS itself can reduce or abolish MEPs (Taylor, Petersen, Butler, & Gandevia, 2002), indicating that CMS activates a similar portion of large corticospinal axons as TMS. Although CMS may be uncomfortable and is not easily acquired in all participants, two main advantages arise when using CMS over other methods testing motoneuronal excitability (e.g., H-reflex). The first is that CMS has a large monosynaptic connection with spinal motoneurons of the upper (Petersen, Taylor, & Gandevia, 2002) and lower (Martin et al., 2008) limbs. The second advantage is that CMS of the corticospinal tract is not subject to presynaptic inhibition (Jackson, Baker, & Fetz, 2006; Nielsen & Petersen, 1994). Taken together, CMS permits a direct assessment of 13 motoneuronal excitability during activity which can greatly aid our understanding of mechanisms leading to exercise-induced fatigue. The neuromuscular junction and muscular contraction: The excitatory and contractile state of the neuromuscular junction and skeletal muscle fiber can be examined with magnetic or electrical PNS (Merton, 1954; Verges et al., 2009). Stimulation intensity is determined by progressively increasing stimulator output until a plateau is obtained in maximal isometric twitch force and/or Mmax amplitude. To ensure the peripheral nerve is adequately stimulated to offset any small alterations in muscle fiber membrane excitability, a supramaximal intensity is typically chosen and often set at 130% of the stimulator intensity which elicits a plateau in twitch force and/or Mmax amplitude. However, while magnetic stimulation may be less painful to the participant, it may not always achieve a supramaximal stimulation due to limits on the stimulator output (Evans, Litchy, & Daube, 1988), thus electrical PNS will be used throughout the studies of this dissertation. The amplitude of the M-wave produced by PNS reflects the summation of action potentials evoked in individual muscle fibers (Bigland-Ritchie, Kukulka, Lippold, & Woods, 1982). In particular, it has been proposed that Mmax reflects sarcolemma excitability and is an indirect measure of Na+/K+ pump function (Hicks, Fenton, Garner, & McComas, 1989; Hicks & McComas, 1989) as the likelihood of presynaptic failure is small (Krnjevic & Miledi, 1958). While the integrity of the sarcolemma is generally maintained during exercise (Bigland-Ritchie et al., 1982; Bigland-Ritchie & Woods, 1984), slight reductions in electrical propagation may be present after exercise (Milner-Brown & Miller, 1986). Reductions in the M-wave amplitude or area are likely associated with an accumulation of K+ in the intracellular space surrounding the axon, impairing the electrochemical gradient 14 necessary for action potential generation (Smith, 1983). Consequently, to account for possible activity-induced changes in muscle fiber potentials, both MEP and CMEP (and possibly voluntary EMG) should be normalized to Mmax (Gandevia et al., 1999; G. Todd, Taylor, & Gandevia, 2003). PNS elicits a brief mechanical response in the target muscle, termed a twitch, which is devoid of central factors and provides information concerning muscle excitationcontraction coupling and contractile kinetics. The magnitude of this twitch is influenced by prior contraction history (postactivation potentiation), a phenomenon explained by the activity-dependent phosphorylation of myosin light chains which sensitizes the actin and myosin filaments to Ca2+ (Palmer & Moore, 1989). Therefore, each twitch should be evoked immediately following a standardized 2-5 s MVC for the evaluation of potentiated twitch force (e.g., quadriceps twitch force Qtw). Interestingly, the degree of potentiation has been previously used to indicate muscle fiber type in vivo (Hamada, Sale, MacDougall, & Tarnopolsky, 2000); however, further corroboration is necessary before this technique can be widely accepted. In addition to single stimuli, pairs or trains of stimuli can be delivered at a range of frequencies to create a force-frequency curve (Allman & Rice, 2004) and examine low-frequency or high-frequency fatigue (Edwards, Hill, Jones, & Merton, 1977). Importantly, the use of PNS to investigate alterations in a muscle's twitch force is invalidated in muscle groups in which the motor nerve innervates both agonists and antagonists (e.g., the brachial plexus nerve of the elbow flexors and extensors (Todd et al., 2003). Testing such muscle groups for contractile function requires direct stimulation of the muscle belly, although nerve stimulation is still necessary to examine the excitability of the corticospinal pathway innervating the muscle group. 15 Assessments of Exercise-Induced Fatigue The assessment of neuromuscular function typically occurs before and again immediately after exercise. The discrepancy between pre- and postexercise measures is then used to quantify fatigue (Amann & Calbet, 2008). While the change in a participant's maximal voluntary contraction (MVC) represents a global measure of muscular fatigue, further analysis of evoked contractile properties and the motor pathway are necessary to identify the components of peripheral and central fatigue. Peripheral fatigue can be quantified by assessing Mmax and Qtw. As stated above, Mmax provides information about the net state of neuromuscular propagation at, and distal to, the stimulation site. On the other hand, the magnitude and rate of rise and fall in the Qtw can indicate impairments in skeletal muscle contractile properties. Specifically, maximal rates of Qtw force development and relaxation, calculated as the greatest positive, or negative derivative (dF/dt), respectively (Bigland-Ritchie, Johansson, Lippold, & Woods, 1983; McNulty, Falland, & Macefield, 2000), provide insight into skeletal muscle excitation-contraction coupling and intracellular Ca2+ handling (Fitts, 2008; Gollnick, Korge, Karpakka, & Saltin, 1991). Additionally, doublets or tetani evoked by 0.5-1s trains of stimuli can be utilized for the determination of high-frequency or low frequency fatigue (Cooper, Edwards, Gibson, & Stokes, 1988; Jones, Bigland-Ritchie, & Edwards, 1979; Metzger & Fitts, 1986). High-frequency fatigue is characterized by a loss of force at high stimulation frequencies (≥80 Hz) and is associated with a failure of the M-wave due to increased K+ and decreased Na+ in the t-tubules (Jones, 1996; Jones et al., 1979). Lowfrequency fatigue is characterized by a greater loss of force at low (≤20 Hz) compared to high (≥50 Hz) stimulation frequencies after fatiguing exercise (especially eccentric 16 contractions which induce muscle damage). The phenomenon can persist for days and is the result of reduced Ca2+ release from the sarcoplasmic reticulum due to impairment of excitation-contraction coupling (Edwards et al., 1977; Jones, 1996). Central fatigue can be quantified by measuring the exercise-induced change in a participant's ability to voluntarily activate a muscle or muscle group. Voluntary muscle activation (VA) can be estimated via the central activation ratio (MVC/MVC + stimulus train; Kent-Braun & Le Blanc, 1996; Place, Maffiuletti, Martin, & Lepers, 2007) and the twitch interpolation technique (Merton, 1954). The current dissertation will examine exercise-induced changes in central fatigue via the twitch interpolation technique. The traditional version of the twitch interpolation technique utilizes PNS during a MVC, which evokes an additional involuntary force termed superimposed twitch (SIT) if muscle activation is submaximal. This additional force is the result of augmenting motor unit recruitment or motoneuron firing rates which were not optimally activated during voluntary effort. Voluntary activation, defined as the force produced voluntarily compared to the maximal possible force (Taylor, 2009), is calculated as follows: 𝑆𝐼𝑇 VA (%) = [1 - (𝑄𝑡𝑤)] • 100 (Belanger & McComas, 1981; Merton, 1954). An exercise-induced increase in the ratio between the SIT and Qtw indicates diminished VA and is interpreted as central fatigue. As an alternative to PNS, VA can be quantified by the interpolated twitch technique with the use of TMS. However, as cortical and motoneuronal excitability are greater during a contraction than at rest (Rothwell et al., 1991), it is inappropriate to use the resting evoked response to TMS for the calculation of VA. To overcome this issue, SITs evoked during brief contractions at 50%, 75%, and 100% MVC can be extrapolated by linear regression ( r ≥ 0.9) to the y-axis, where the y-intercept 17 represents the estimated resting twitch (Todd et al., 2003). In combination with the TMS evoked SIT during the MVC, the estimated resting twitch is applied to the above calculation for VA. Although this method has been found to be reliable in the upper (Todd et al., 2003) and lower (Goodall, Romer, & Ross, 2009; Sidhu, Bentley, & Carroll, 2009) limbs, there is a greater reliance of assumptions for the calculation of VA via TMS compared to PNS, thus the PNS methodology will be used to measure VA in this dissertation. However, it is important to remember that VA is an estimation in both methods and subject to methodological fallacies (for review see de Haan, Gerrits, & de Ruiter, 2009; Taylor, 2009). In addition to VA, exercise-induced changes in EMG (as normalized to Mmax or maximal preexercise EMG obtained during a MVC or the first minute of exercise) have been used as an estimate of central fatigue (Amann & Dempsey, 2008; Bigland-Ritchie, Jones, Hosking, & Edwards, 1978; Loscher, Cresswell, & Thorstensson, 1996a). However, it is important to note that EMG can decline while force is maintained during isometric exercise (Bigland-Ritchie et al., 1983), a characteristic described by the muscle wisdom theory (Marsden, Meadows, & Merton, 1983) in which optimal force production occurs at lower motor unit firings rates during fatiguing exercise (Jones et al., 1979). Therefore, in addition to other methodological considerations (Farina, Merletti, & Enoka, 2004), EMG by itself may not provide a clear indication of central fatigue. Muscle activation is dependent on descending neural input reaching the skeletal muscle to initiate contraction. Alterations in the efficacy of the corticospinal pathway to relay neural drive to the muscle therefore play a significant role in the development of central fatigue (Klass, Levenez, Enoka, & Duchateau, 2008; Martin, Smith, Butler, 18 Gandevia, & Taylor, 2006; Petersen et al., 2003). To this extent, the excitability of the motor cortex, alpha motoneurons, and muscle fiber membrane can be investigated. The magnitude of the MEP can quantify activity-induced alterations within the corticospinal pathway to a target muscle (Day et al., 1989; Rothwell et al., 1991). Although exerciseinduced alterations in MEP provide information regarding the efficacy of the entire corticospinal pathway, it is not possible to decipher between cortical, spinal, or peripheral contributions by using TMS only. In order to circumvent this problem, the use of subcortical stimulation is required. CMEPs can reveal alterations in the excitability of the spinal motoneurons and muscle fibers, so, when expressed as a ratio with MEPs (i.e., MEP/CMEP), the normalized MEP provides insight into the excitability of motor cortical output cells (Martin et al., 2008; Taylor, 2006; Taylor & Gandevia, 2004). The comparison of MEPs to CMEPs is made possible by the fact that CMS activates largely the same pathway as TMS (Martin et al., 2008; Taylor et al., 2002; see also above). In addition to the MEP/CMEP ratio, motor cortical excitability can also be examined via changes in the silent period, SICI, LICI, and ICF, as described above. It should be noted that changes in the excitability of the corticospinal pathway are transient, returning to baseline within seconds or minutes after the cessation of exercise indicating the importance for quick measurements (Taylor et al., 1996). Aim for Study 1 The descending neural drive from higher brain centers needed to evoke skeletal muscle contraction is conducted primarily via the corticospinal motor pathway, including 19 the motor cortex and spinal motoneurons. Maintaining or enhancing the functional integrity of these neuronal structures during physical activity is an important prerequisite for the CNS to optimally activate the muscles necessary for a given task. Alterations in the efficacy of the corticospinal pathway to relay neural signals (i.e., changes in corticospinal excitability) may influence the development of central fatigue and the control of voluntary movements (Klass et al., 2008; Martin, Smith, et al., 2006; Petersen et al., 2003). For example, a decrease in corticospinal excitability during exercise would require an increased input from higher brain centers to maintain a given power output (Di Lazzaro et al., 1998a; Martin, Smith, et al., 2006; Mazzocchio, Rothwell, Day, & Thompson, 1994). Consequently, it is important to understand how exercise may influence the integrity of this motor pathway. Increases in muscle activation and the associated electromyographic (EMG) activity (Alkner, Tesch, & Berg, 2000), have been demonstrated to enhance the excitability of the corticospinal pathway during isometric contractions of the upper and, to a lesser extent, lower limb (Martin et al., 2008; Taylor, Allen, Butler, & Gandevia, 1997; Todd et al., 2003). Specifically, MEPs and CMEPs progressively grow with increasing isometric force of the lower limb up to 50% MVC, after which further increases in force result in an unchanged (Oya, Hoffman, & Cresswell, 2008) or diminished (Goodall et al., 2009; Sidhu et al., 2009) corticospinal excitability of the lower limb. However, while a growing number of investigations examine corticospinal excitability during rhythmic exercise (Carroll, Baldwin, Collins, & Zehr, 2006; Sidhu, Cresswell, & Carroll, 2012; Sidhu, Hoffman, Cresswell, & Carroll, 2012), the influence of increasing muscle activation during rhythmic exercise is unknown. This is of particular importance as exercise, such as fatiguing 20 constant-load cycling, necessitates a progressive increase in EMG throughout the task (Amann et al., 2011). Therefore, the purpose of Study 1 is to describe the influence of exercise intensity on cortical and spinal excitability of the knee extensors during both nonfatiguing single isometric knee-extensor contractions and dynamic cycling. Furthermore, the alterations in corticospinal excitability for a given change in muscle activation will be examined across exercise modalities in order to determine if there are task-specific variations. Aim for Study 2 In addition to various neuromuscular alterations (Allman & Rice, 2002; Power, Dalton, & Rice, 2013), aging is associated with decrements in the cardiovascular and respiratory systems. Age-related impairments in limb blood flow (Donato et al., 2006; Proctor et al., 1998), cardiac function (Ferrari, Radaelli, & Centola, 2003), and ventilatory work (Jordan & Jerome, 2004) have the potential to diminish systemic oxygen delivery, a key determinant of the development of muscle fatigue during physical activity (Amann et al., 2006). Given, compared to single-joint exercise, the larger requirements and involvement of the cardiorespiratory system during whole body exercise (Esposito, Mathieu-Costello, Shabetai, Wagner, & Richardson, 2010; Rossman, Venturelli, McDaniel, Amann, & Richardson, 2012), there is a greater potential for the cardiovascular and ventilatory systems to expedite the development of fatigue during whole body exercise in old compared to young individuals. However, to date there have been no investigations focusing on the impact of age on the development of neuromuscular fatigue during whole 21 body exercise. Our current understanding of the impact of age on the development of fatigue during exercise is convoluted by the wide range of methodologies employed and muscle groups investigated (Avin & Law, 2011; Christie, Snook, & Kent-Braun, 2011). Indeed, studies have attributed a greater (Hunter, Critchlow, & Enoka, 2005; Lanza, Russ, & KentBraun, 2004; Rawson, 2010), lesser (Dalton, Power, Paturel, & Rice, 2015; Lindstrom, Karlsson, & Lexell, 2006; McNeil & Rice, 2007), or similar (Callahan, Foulis, & KentBraun, 2009; Christie & Kamen, 2009; Lindstrom, Lexell, Gerdle, and Downham, 1997) fatigue resistance to older participants when compared to their younger counterparts. Additionally, the majority of the literature in this field examines the fatigability associated with age during single-joint exercise including a small muscle mass at relative exercise intensities (Avin & Law, 2011). However, exercise including a small muscle mass engages the cardiopulmonary system to a much lower degree compared to whole body exercise including a large muscle mass. These studies therefore neglect the known cardiovascular and pulmonary alterations associated with aging which may impede oxygen delivery to the working muscle (Ferrari et al., 2003; Lawrenson et al., 2003), a major determinant of exercise-induced fatigue (Amann & Calbet, 2008). Due to these restrictions, the current literature may, likely unintentionally, offset any age-related advantage or disadvantage. The purpose of Study 2 is to employ two protocols (single-leg dynamic and whole body cycling exercise) to comprehensively investigate the effect of aging on the development of fatigue during exercise. By employing both relative and absolute exercise intensities for each protocol, we will be able to comprehensively investigate the impact of age on the development of fatigue during exercise. 22 Aim for Study 3 In 2009, over 5.8 million people in the U.S. had been diagnosed with heart failure (HF), with greater than 550,000 new cases identified each year (Hunt et al., 2009). Traditionally, HF has been associated with a reduced ejection fraction (HFrEF). However, it is now recognized that approximately half of the patients hospitalized with HF are characterized by a preserved ejection fraction (≥50%; HFpEF; Owan et al., 2006). Similar to HFrEF, HFpEF patients suffer from significant impairments in exercise tolerance and quality of life (Kitzman et al., 2002). However, cardiac function has been reported to be dissociated from improvements in exercise tolerance as endurance training in HFpEF has been documented to improve exercise tolerance without changes in cardiac function (Kitzman, Brubaker, Morgan, Stewart, & Little, 2010). Subsequently, a less "cardiocentric" approach in explaining the exercise intolerance in this population has been recently gaining support (Dhakal et al., 2015; Haykowsky, Tomczak, Scott, Paterson, & Kitzman, 2015; W. J. Paulus & Tschope, 2013). Indeed, it is now known that HFpEF patients demonstrate significant impairments in the periphery including limitations in limb blood flow (Lee et al., 2016), oxygen extraction by the skeletal muscle (Bhella et al., 2011; Dhakal et al., 2015), and a shift from oxidative Type I fibers to less oxidative Type II fibers (Kitzman et al., 2014). As these factors contribute to the delivery and utilization of oxygen by the contracting skeletal muscle, a primary determinant of muscle fatigue (Amann & Calbet, 2008), exercise intolerance in this population may be the result of an exacerbated development of fatigue. However, there are currently no studies which have investigated the development of fatigue during physical activity in patients with HFpEF. The purpose of Study 3 is to investigate the impact of HFpEF on the development of peripheral and 23 central fatigue during small muscle mass exercise. Specifically, the development of fatigue during intermittent isometric and dynamic knee extensions in patients with HFpEF will be compared to that in age-matched healthy controls. Summary of Specific Aims of Dissertation Older individuals (≥65 years) and patients with HFpEF are, although to different degrees, characterized by systemic and neuromuscular deficiencies which may predispose these populations to develop fatigue during exercise at a faster rate compared to healthy young individuals (Avin & Law, 2011; B. C. Clark & Manini, 2010; Hopkinson et al., 2013; Kitzman et al., 2014). Physical activity may therefore lead to premature fatigue and consequently exercise intolerance in these populations (Amann et al., 2011; Borlaug et al., 2006; D. J. Clark et al., 2011; Dalton et al., 2015; Wilson, Martin, Schwartz, & Ferraro, 1984). As low fatigue resistance and exercise intolerance have far reaching significant negative consequences on an individual's quality of life (Pandey et al., 2015), the purpose of this dissertation is to gain a more comprehensive understanding of the development of fatigue during physical activity in both healthy older individuals and patients with HFpEF. Therefore, the aims of the current dissertation are to examine: 1) The influence of neural drive on the excitability of the corticospinal pathway. 2) The impact of age on the development of fatigue in whole body and single-joint exercise 3) The impact of HFpEF the development of fatigue during small muscle mass, single-joint exercise. 24 References Adam, A., & De Luca, C. J. (2003). Recruitment order of motor units in human vastus lateralis muscle is maintained during fatiguing contractions. Journal of Neurophysiology, 90(5), 2919-2927. doi: 10.1152/jn.00179.2003 Alkner, B. A., Tesch, P. A., & Berg, H. E. (2000). 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Transcranial magnetic stimulation (TMS), cervicomedullary stimulation (CMS), and peripheral nerve stimulation (PNS) create an electrical signal transmitted to the skeletal muscle. This signal can be quantified via surface electromyography (EMG, blue squares), where the latency between the stimulus and response will depend on the location of stimulation (e.g., A,B,C). CHAPTER 2 INTENSITY-DEPENDENT ALTERATIONS IN THE EXCITABILITY OF CORTICAL AND SPINAL PROJECTIONS TO THE KNEE EXTENSORS DURING ISOMETRIC AND LOCOMOTOR EXERCISE Reprinted with permission from the American Physiological Society. J. C. Weavil, S. K. Sidhu, T. S. Mangum, R. S. Richardson, and M. Amann. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 308: R998-R1007, 20 41 Abstract Introduction 42 Methods 43 44 Results 45 46 Discussion 47 48 49 Grants Disclosures Author contributions References 50 CHAPTER 3 THE IMPACT OF AGE ON THE DEVELOPMENT OF FATIGUE DURING LARGE AND SMALL MUSCLE MASS EXERCISE 52 Abstract This study examined the impact of aging on neuromuscular fatigue following cycling (CYC; large muscle mass) and single-leg knee-extension (KE; small muscle mass) exercise. Eight young (25 ± 1years) and older (70 ± 1years) participants performed CYC and KE to exhaustion at a given relative (80% Wpeak) and absolute intensity. Peripheraland central-fatigue were quantified via the pre/post-exercise changes in quadriceps twitch torque (∆Qtw, electrical femoral nerve stimulation) and voluntary activation (∆VA), respectively. Absolute exercise intensity: In both modalities, the old demonstrated a 23% greater fall in ΔQtw and a 4% greater fall in ∆VA compared to the young individuals (P < 0.05). Relative exercise intensity: Although the young participants performed 33% and 77% more work during KE and CYC, respectively, time to task-failure was similar between groups in both modalities (~9.5min). While peripheral-fatigue was similar between groups (ΔQtw ~45%; P > 0.5), end-exercise central-fatigue was larger in the young (∆VA +4%). These findings reflect an impaired neuromuscular fatigue resistance during exercise at a given absolute intensity in older individuals, but suggest that this population develops, compared to the young, less central-fatigue during exercise performed at the same relative intensity. Furthermore, active muscle mass has little influence on the age-related discrepancy in the exercise-induced development of neuromuscular fatigue. Introduction Investigations addressing the effect of aging on the development of neuromuscular fatigue have traditionally only focused on single-joint exercise (Avin & Law, 2011), a 53 modality which is not limited by central cardiopulmonary constraints (Andersen & Saltin, 1985). However, exercise involving a large muscle mass, such as whole body cycling, is associated with substantial cardiopulmonary responses which are unfavorably altered with aging. For example, age-related constraints in cardiovascular function (Donato et al., 2006; Ferrari, Radaelli, & Centola, 2003; Wray & Richardson, 2006) and ventilatory work (Jordan & Jerome, 2004) have the potential to limit limb blood flow and oxygen delivery during intense exercise (Dempsey, Amann, Romer, & Miller, 2008; Jordan & Jerome, 2004) and therefore exacerbate the development of neuromuscular fatigue (Dempsey et al., 2008; Esposito, Mathieu-Costello, Shabetai, Wagner, & Richardson, 2010; Harms et al., 1997). Consequently, by only utilizing single-joint exercise, the current literature may unintentionally offset the influence of age on the development of fatigue by negating agerelated impairments of the cardiopulmonary system. To fully appreciate the impact of aging on the development of fatigue, it is pertinent to examine both small and large muscle mass exercise. Exercise-induced neuromuscular fatigue is defined as a reversible decrease in the torque or power generating capacity of a muscle or muscle group (Gandevia, 2001). This temporary impairment is determined by a decrease in neural activation of the muscle (i.e., central fatigue) and/or biochemical changes at or distal to the neuromuscular junction that cause an attenuated contractile response to neural input (i.e., peripheral fatigue; BiglandRitchie, Jones, Hosking, & Edwards, 1978). Notably, as muscle activation is critically dependent on descending neural drive, the integrity of the motor pathway linking the brain with the exercising skeletal muscle, termed the corticospinal pathway (including the motor cortex and spinal motoneuron), is important. To monitor alterations in the excitability of 54 the corticospinal pathway of a particular muscle, transcranial magnetic stimulation, a noninvasive method that evokes short-latency electromyographic responses, termed motorevoked potentials, can be used (Day et al., 1989; Rothwell, Thompson, Day, Boyd, & Marsden, 1991). Furthermore, in order to decipher alterations in corticospinal excitability at a segmental level, cervicomedullary stimulation of corticospinal axons can be employed to differentiate alterations at the motoneuronal and, by extension, the motor cortical level (Taylor & Gandevia, 2004). The aim of this study was to elucidate the impact of age on the development of fatigue during both large (cycling) and small (rhythmic single-joint knee extension, KE) muscle mass exercise. Briefly, KE has a similar force profile as cycling exercise and is characterized by a cardiac and pulmonary reserve even at maximal exercise (Esposito et al., 2010), thereby reducing cardiopulmonary constraints on limb blood flow and O2 delivery which are present during cycling exercise. Furthermore, the vast majority of literature has focused on relative intensity exercise, which may obscure the practical (i.e., pertinent to activities of daily living) age-related influence on the development of fatigue (Avin & Law, 2011). Therefore, each modality was performed at both absolute and relative exercise intensities. It was hypothesized that with exercise performed at a given relative intensity, aging would exacerbate the development of fatigue during cycling, but has no influence on end-exercise neuromuscular fatigue during KE (reducing the age-related influence of the cardiopulmonary system). Additionally, we hypothesized that older individuals incur greater neuromuscular fatigue in both modalities when the task is performed at a given absolute intensity. 55 Methods Participants Sixteen healthy, recreationally active males (8 young and 8 older) volunteered for this study (Table 3.1). Written, informed consent was obtained from all participants before their inclusion in the study. The Institutional Review Boards of the University of Utah and the Salt Lake City Veterans Affairs Medical Center approved all protocols. Experimental Procedures During preliminary visits, anthropometric measurements were collected and participants were familiarized with all experimental testing procedures. All exercise sessions followed a standardized, task-specific warm-up and were separated by at least 24 hours. Quadriceps fatigue was quantified by the pre- to postexercise decrease in neuromuscular function (i.e., peak torque obtained during maximal voluntary contractions [MVC], evoked twitch torque, and voluntary muscle activation). To quantify exerciseinduced changes in corticospinal excitability, participants received 3 stimulation sets before and immediately after exercise. Each stimulation set consisted of three transcranial magnetic stimulations (TMS), one cervicomedullary stimulation (CMS), and one femoral nerve stimulation (FNS). The stimulations were randomized and triggered during a constant quadriceps contraction equating to 20% of the electromyographic (EMG) activity recorded during preexercise MVCs (Figure 3.1). Protocol A - Dynamic single-leg knee extension: The experimental protocol is depicted in Figure 3.1 A. At the beginning of all exercise sessions and prior to baseline 56 neuromuscular measures, participants performed a 1-minute unloaded warm-up on a custom-made knee extensor ergometer. The first preliminary visit consisted of a maximal incremental single-leg knee extensor exercise test (range of motion: 90-170° of knee angle). Subjects were asked to maintain a consistent cadence at 60 rpm throughout all testing procedures. Peak power output (Wpeak) and oxygen consumption (V̇O2peak) were determined by increasing the work rate from an unloaded state by 5-10 W·min-1 until task failure (rpm drops below 50 for >5 s despite verbal encouragement). Wpeak represents the workload during the last stage completed while V̇O2peak represents the average O2 consumption during the last minute of exercise. During the 2nd and 3rd visit, participants performed familiarization trials at 80% Wpeak to task failure (Tlim). During the 4th visit, considered the experimental day, participants performed the 80% Tlim exercise and exercise-induced neuromuscular quadriceps fatigue and changes in corticospinal excitability were assessed. Protocol B - Leg cycling exercise: The experimental protocol is depicted in Figure 3.1 B. Prior to all exercise sessions, participants performed a standardized warm-up on a cycle ergometer (Velotron, Elite model, Racer Mate, Seattle, WA) at 25, 50, and 75 W for 3 minutes each. During the 1st visit, an incremental cycling test to task failure (starting at 20 W and adding 25 W/min) was performed by all participants. Wpeak and VȮ2peak were determined as described above. The goal of visits 2-4 were similar to those in Protocol A. 57 General Instrumentation Physical activity level: Once instructed on proper operating procedures, participants wore an accelerometer (GTIM; Actigraph, Pensacola, FL) for 10 consecutive days. Total daily physical activity were averaged and reported as steps per day and physical activity counts per minute. The counts per minute will be subsequently categorized into sedentary, low, moderate, and vigorous intensities using commercially available software (Actilife, Actigraph, Pensacola, FL). Physical activity level: Once instructed on proper operating procedures, participants wore an accelerometer (GTIM; Actigraph, Pensacola, FL) for 10 consecutive days. Total daily physical activity were averaged and reported as steps per day and physical activity counts per minute. The counts per minute was subsequently categorized into sedentary, low, moderate, and vigorous intensities using commercially available software (Actilife, Actigraph, Pensacola, FL). Determination of quadriceps muscle mass: Quadriceps muscle volume was estimated via anthropometric measurements as described by Jones and Person (Jones & Pearson, 1969) while applying a recommended correction factor for the determination of quadriceps muscle mass (Andersen & Saltin, 1985). Electromyography: The EMG signals were recorded by surface electrodes (AgAgCl, 10-mm diameter) placed on the muscle belly and tendon of the vastus lateralis (VL) in a monopolar configuration. Prior to electrode placement, the skin was lightly abraded with fine sandpaper and cleaned with an alcohol swab. EMG signals were amplified 1000 times (Neurolog Systems, Digitimer Ltd., Welwyn Garden City, Hertfordshire, England, UK), band-pass filtered (20-1000 Hz; NL-844, Digitimer Ltd), and analog to digitally 58 converted at a sampling rate of 2000 Hz using a 16-bit Micro 1401 mk-II and Spike 2 data collection software (Cambridge Electronic Design Ltd, Cambridgeshire, England, UK) running custom written scripts. The right knee angle was monitored continuously during dynamic knee extensor and cycling exercise. During a brief (~45 s) bout of exercise at the experimental workload, the knee angle at which the peak EMG burst of the VL occurs (i.e., the centering point) was determined. For EMG analysis during exercise, 10 seconds of rectified EMG waveforms were overlaid around the crank angle centering point and averaged (Weavil, Sidhu, Mangum, Richardson, & Amann, 2015). EMG was averaged over a 100 ms period, that is, 50 ms before and 50 ms after the centering point, to provide the average knee extensor EMG at each time point. EMG was normalized to that obtained during the first minute of exercise. Neuromuscular quadriceps function and torque acquisition: To examine exerciseinduced fatigue, measures of neuromuscular quadriceps function of the dominant (right) leg were assessed before and immediately (37 ± 2 s) after exercise. For KE, participants were seated upright in the knee extensor ergometer with their hip and knee flexed at 120° and 90°, respectively. For cycling exercise, participants were moved to a custom-made chair with their hip and knee flexed at 120° and 90°, respectively. The time between the end of exercise and the beginning of the postexercise assessment of neuromuscular function was similar between KE and cycling exercise (~30s). A noncompliant cuff was attached to a calibrated load cell (MLP 300; Transducer Techniques, Inc. Temecula, CA, USA) 2-3 cm superior to the lateral malleolus. Cuff position remained secured throughout each session. The assessment of quadriceps function included three MVCs, over which a FNS was elicited. If muscular activation was suboptimal during the MVC, the FNS would 59 evoke a superimposed twitch (SIT). Following each MVC, another FNS stimulation was initiated to evoke a resting potentiated quadriceps twitch (Qtw). Voluntary quadriceps activation (VA) was calculated as: VA = [1-(SIT/Qtw)]*100 (Belanger & McComas, 1981; Merton, 1954). Exercise-induced peripheral and central fatigue was quantified as the percent reduction of the averaged Qtw and VA, respectively, from before to after exercise. Furthermore, the maximal rate of torque development (MRTD, calculated as the highest positive derivative of the torque during a 10-ms interval) and peak relaxation rate (PRR, the highest negative derivative of the torque during a 10-ms interval) were analyzed for each Qtw. FNS: In all protocols, the motor nerve was stimulated with the anode placed between the greater trochanter and the iliac crest and the cathode placed over the femoral nerve in the femoral triangle. Optimal position (i.e., greatest twitch force) for the stimulating electrode was determined by delivering low intensity single pulse stimuli (200 µs pulse width; 100-150 mA) via a movable cathode probe and a constant current stimulator (voltage range: 100-400V; Model DS7AH, Digitimer Ltd., Welwyn Garden City, Hertfordshire, England, UK). Once located, the cathode was fixed and remained in this position for the remainder of the session. Thereafter, stimulation intensity was increased by 20 mA increments until the size of the evoked twitch and compound muscle action potential (M-wave) demonstrated no further increase (i.e., maximal M-wave, Mmax) at rest further confirmed during a 50% MVC. Stimulation intensity was set at 130% of Mmax intensity and kept constant throughout the testing session (Young: 338 ± 10 mA; Old: 460 ± 57 mA). CMS: The corticospinal pathway was electrically stimulated at the level of the 60 cervicomedullary junction. Self-adhesive electrodes were placed on the grooves behind the mastoid processes with the cathode placed on the left (100 µs pulse width, D-185 mark IIa, Digitimer Ltd., Welwyn Garden City, Hertfordshire, England, UK; Ugawa, Rothwell, Day, Thompson, & Marsden, 1991). Stimulator intensity was set to achieve a cervicomedullary motor-evoked potential (CMEP) of approximately 30% Mmax during a constant quadriceps contraction at an intensity corresponding to 20% of the EMG obtained during a quadriceps MVC (Young: 509 ± 54 V; Old: 520 ± 40 V). This procedure provides room for either an exercise-induced increase or decline in corticospinal responses (Sidhu, Cresswell, & Carroll, 2012). TMS: Initially, stimuli were delivered over the vertex of the motor cortex (left hemisphere, approximately 2-3 cm lateral of the vertex) using a concave double-cone coil (Magstim 200; Magstim Co. Ltd, Whitland, UK) to elicit MEPs in the quadriceps during all protocols. Optimal positioning of the TMS coil was determined prior to the experiment and marked on the scalp for accurate placement throughout the study. In order to test a similar portion of the motoneuronal pool, the stimulator intensity was set to evoke MEPs of similar size to the CMEP obtained during a constant quadriceps contraction at an intensity corresponding to 20% of the EMG obtained during a quadriceps MVC (Sidhu et al., 2012; Young: 56 ± 4% of stimulator output; Old: 53 ± 4% of stimulator output). If CMS was not tolerated (4 of 8 older individuals in the Protocol B), TMS was set at approximately 30% Mmax. 61 Protocol Specific Instrumentation Protocol A - Dynamic single-leg knee extension: Ventilation, pulmonary gas exchange, and cardiovascular measures: Ventilation and pulmonary gas exchange was measured with a metabolic cart (Innocor, Innovision, Odense, Denmark). Heart rate was measured via the R-R interval derived from a 12-lead electrocardiogram acquired with a data acquisition system (Spike 2, Cambridge Electronic Design Ltd, Cambridgeshire, England, UK). Mean arterial pressure (MAP) was determined via a Finometer utilizing finger photoplethysmography (Finapres Medical Systems, Amsterdam, The Netherlands). Stroke volume (SV) was estimated via beat-by-beat assessment from the pressure waveform using the Modelflow method (Beatscope, version 1.1, Finapress Medical Systems). Cardiac output (CO) was calculated as the product of HR and SV. Systemic vascular conductance (SVC) was calculated as CO/MAP. Participant's rating of perceived exertion (RPE) was obtained at the end of each minute during exercise (Borg, 1982). Leg blood flow: Femoral blood flow (FBF) measurements were collected at rest and during exercise (for visits 1 and 4 of Protocol A) as previously described (Wray & Richardson, 2006). Briefly, a Logic 7 ultrasound (General Electric Medical Systems, Milwaukee, WI) was used for measurements of vessel diameter and blood velocity of the common femoral artery (CFA), proximal to the bifurcation of the superficial and deep femoral artery. Using CFA diameter and mean blood velocity (Vmean), FBF (mL/min) was calculated as: Vmean·π·(vessel diameter/2)2·60. The ultrasound probe was positioned to maintain an insonation angle of 60° or less during all blood velocity measurements. Leg vascular conductance (LVC) was calculated as FBF/MAP. Protocol B - Leg cycling exercise: Ventilation, pulmonary gas exchange, 62 cardiovascular measures, and EMG analysis: Ventilation, pulmonary gas exchange, HR, and RPE were measured as described in Protocol A. Based on the time to task failure during the last practice trials, CO was measured at 90% of the Tlim completion time via an inert gas rebreathing technique (Innocor, Innovision, Odense, Denmark) (Peyton & Thompson, 2004). EMG analysis was performed during cycling exercise as described in Protocol A. Data Analysis All data were stored and analyzed offline using Spike2 data acquisition software. The area for each evoked response (MEP, CMEP, and Mmax) was measured and averaged over the three sets of stimulations. To account for potential changes within the muscle, MEPs and CMEPs were normalized to Mmax. To isolate alterations in the excitability of the motor cortex, MEP was normalized to CMEP (Martin, Weerakkody, Gandevia, & Taylor, 2008). All data were reported as mean ± standard error of the mean (SE). Statistical Analysis A priori comparisons were performed between the young and old groups for both the relative and absolute condition. Two-way mixed model ANOVAs (group by modality) were performed to determine if there is a significant effect of age and exercise modality on exercise-induced neuromuscular fatigue (i.e., MVC, Qtw, and VA). Additional two-way mixed model ANOVAs (group by workload and group by time) were performed for the comparison of LBF during the single-leg dynamic maximal and 63 Tlim trials, respectively. If a main effect was identified, a Holm-Sidak post hoc test was performed to determine where differences occurred. Additionally, Student's t tests were employed to compare descriptive characteristics, baseline neuromuscular function, preand postexercise changes in twitch mechanics (MRTD and PRR), corticospinal excitability (Mmax, MEP, CMEP, and MEP/CEMP), and end-exercise (last minute) metabolic and ventilatory data. Alpha was set at 0.05. Results Subject Characteristics Subject characteristics and baseline neuromuscular function are presented in Table 3.1. Young and older participants were well matched for physical activity, anthropometric measures, and hematologic characteristics. Protocol A: Dynamic Knee Extension Incremental exercise test: Knee extensor Wpeak was higher in the young than the older group (68 ± 3 W and 55 ± 2 W, respectively; P < 0.01). Additionally, although FBF was higher at peak exercise in young compared to old (4217 ± 191 ml/min vs. 3577 ± 223 ml/min, P < 0.05), there was no difference during unloaded KE (~1460 ml/min), 10 W (~1830 ml/min), 20 W (~2300 ml/min), and 40 W (~2910 ml/min; group: F1,14 = 2.61, P = 0.13, time: F1,14 = 140, P < 0.001). Relative exercise intensity: Calculated coefficients of variation for the time to task 64 failure were 11% and 8% in the young and older participants. Although time to task failure was similar between young and older participants (650 ± 62 s and 601 ± 56 s, respectively; P = 0.57), the young completed more work during the task (36 ± 4 kJ vs. 27 ± 3 kJ, P < 0.05). VL-EMG increased similarly (~90% from first minute) throughout the exercise in both groups (P = 0.61). FBF (group: F1,14 = 5.3, P < 0.05; time: F1,14 = 27.4, P < 0.05) and LVC (group: F1,14 = 5.5, P < 0.05; time: F1,14 = 3.9, P < 0.05) increased throughout Tlim in both groups, but were greater in the young (Figure 3.2). Other cardiovascular and metabolic data are shown in Table 3.2. The exercise-induced changes in neuromuscular function are reflected in Figure 3.3. In addition, the exercise-induced decrease in MRTD (young: -52 ± 6 % vs. old: -49 ± 7 %; P = 0.69) and PRR (young: -50 ± 6 % vs. old: -40 ± 4 %; P = 0.20) were similar in both groups. Pre- and postexercise Mmax were similar in both the young (30.0 ± 4.0 μV·s vs. 30.9 ± 3.4 μV·s, P = 0.28) and old (30.7 ± 5.2 μV·s vs. 30.8 ± 5.1 μV·s, P = 0.87). Furthermore, normalized MEP (~36% Mmax, P = 0.62), normalized CMEP (~33% Mmax, P = 0.75), and MEP/CMEP (~113 %, P = 0.65) were unchanged from baseline after exercise in the young. Similarly, normalized MEP (~34% Mmax, P = 0.47), normalized CMEP (~27% Mmax, P = 0.68), and MEP/CMEP (~135%, P = 0.79) were unchanged from baseline after exercise in the old. Absolute exercise intensity: By design, KE exercise time and work were matched between groups. VL EMG in the young remained unchanged throughout the exercise (P = 0.11) while it increased by 100 ± 32% (P < 0.05) in the old. There was an interaction effect for FBF with age (interaction: F1,6 = 3.8, P < 0.05) (Figure 3.2). In contrast, LVC was similar between groups (group: F1,14 = 0.3, P = 0.59). Other cardiovascular and metabolic 65 data are shown in Table 3.2. The exercise-induced changes in neuromuscular function are reflected in Figure 3.3. Mmax was unaltered by exercise in the young (~33 μV·s, P = 0.66). Furthermore, normalized MEP (~32% Mmax, P = 0.93), normalized CMEP (~31% Mmax, P = 0.82), and MEP/CMEP (~113%, P = 0.75) were unchanged from pre- to postexercise in the young and not different from the old (P > 0.20). Protocol B: Cycling Exercise Incremental exercise test: Wpeak was higher in the young compared to the old (309 ± 25 W and 195 ± 10 W; P < 0.01). Additionally, V̇O2peak was significantly greater in the young compared to the old (3.64 ± 0.26 L/min vs. 2.26 ± 0.14 L/min; P < 0.05) Relative exercise intensity: Calculated coefficients of variation reflect acceptable between-day variability for time to task failure in both young (10%) and older (8%) participants. Although time to task failure was similar between young and old (545 ± 40 s vs. 506 ± 29 s, respectively; P = 0.44), the young completed more total work during the exercise (140 ± 24 kJ vs. 79 ± 7 kJ, P < 0.05). VL-EMG increased similarly (~30% from the first minute) throughout the exercise in both groups (P = 0.68). Other cardiovascular and metabolic data are shown in Table 3.2. The exercise-induced changes in neuromuscular function are reflected in Figure 3.3. Additionally, MRTD (young: -39 ± 10 % vs. old:-39 ± 9 %; P = 0.99) and PRR (young: -39 ± 9 % vs. old: -34 ± 13 %; P = 0.72) were diminished equivocally between groups. However, there were no age-related effects for exercise modality (cycling vs. KE) in MVC, Qtw, or VA (interaction: F1,14 < 1.51, P > 0.30). 66 Pre- and postexercise Mmax were similar in both the young (~48 μV·s, P = 0.85) and old (~41 μV·s, P = 0.54). Furthermore, normalized MEP (~28% Mmax, P = 0.81), CMEP (~26% Mmax, P = 0.71), and MEP/CMEP (~110%, P = 0.41) were unchanged from baseline after exercise in the young. Similarly, normalized MEP (~29% Mmax, P = 0.40), normalized CMEP (~27% Mmax, P = 0.88), and MEP/CMEP (~102%, P = 0.94) were unchanged from baseline after exercise in the old and not different from the young (P > 0.41). Absolute exercise intensity: By design, cycling exercise time and work was matched between groups. VL-EMG in the young remained unchanged throughout exercise (P = 0.21) but increased by 100 ± 32% from the first minute (P < 0.05) in the old. Other cardiovascular and metabolic data are shown in Table 3.2. The exercise-induced changes in neuromuscular function are reflected in Figure 3.3. Furthermore, in contrast to the old, neither MRTD nor PRR (P > 0.22) were altered by the exercise in the young group. Pre- and postexercise Mmax were similar in the young group (~54 μV·s, P = 0.27). Furthermore, normalized MEP (~31% Mmax, P = 0.90), normalized CMEP (~33% Mmax, P = 0.88), and MEP/CMEP (~106%, P = 0.84) were unchanged from baseline after exercise in the young and not different from the old (P > 0.35). Discussion This study sought to elucidate age-related differences in the development of fatigue during whole body and single joint exercise. Independent of the exercise modality, when matched for absolute intensity and exercise time, old individuals developed significantly 67 more central and peripheral fatigue than their younger counterparts. Furthermore, following exercise at the same relative intensity and for a comparable time, peripheral fatigue was similar between groups while central fatigue was lower in the elderly. However, it is critical to consider that young individuals also performed significantly more work during this comparison. These findings suggest that aging compromises fatigue resistance during exercise. Furthermore, in contrast to exercise modality (i.e., amount of active muscle mass), exercise intensity (i.e., absolute vs. relative) plays a large role in highlighting the influence of aging on fatigue resistance during rhythmic, submaximal physical activity. Whole Body Versus Single-Joint Exercise Aging is associated with impairments in the cardiopulmonary system (Jordan & Jerome, 2004) which may impede exercising limb blood flow and O2 delivery during cycling exercise more in older compared to younger individuals (Dempsey et al., 2008; Harms et al., 1997). Dynamic KE exercise reduces these confounding influences (Esposito et al., 2010; Rossman, Venturelli, McDaniel, Amann, & Richardson, 2012) and the development of fatigue may only be minimally influenced by the pulmonary system and central hemodynamics (Esposito et al., 2010; Rossman et al., 2012). However, as the impact of aging on the development of fatigue was similar in KE and cycling exercise (no significant age by modality interaction; Figure 3.3), it might be postulated that the aging cardiopulmonary system has little influence on the greater fatigue in older individuals, even during a task with a relatively high cardiopulmonary demand. Additional studies using, for 68 example, assist ventilation to unload the pulmonary system during exercise are needed to further investigate this hypothesis. Development of Fatigue During Exercise at a Given Absolute Intensity This study investigated the development of fatigue in young and older participants during exercise at a given submaximal power output (45 W, corresponding to ~60% and ~80% of Wpeak in young and old, respectively) and for the same duration (~10 min). Both central and peripheral fatigue were significantly greater following KE and cycling exercise in the old compared to the young participants (Figure 3.3). Age-related differences in exercising limb blood flow and likely O2 delivery, a key determinant of the development of fatigue during muscle contractions (Amann & Calbet, 2008), might be excluded as a factor contributing to this discrepancy. Specifically, both the hyperemic response to KE and [Hb] (therefore likely arterial oxygenation (Amann et al., 2014; Esposito et al., 2010)) were comparable between groups (Figure 3.2, Table 3.1). Although this contradicts some (Donato et al., 2006; Wray & Richardson, 2006), but not all (Proctor et al., 2003), previous studies, the observed similarity is not unusual particularly as our participants were engaged in regular physical activities, a factor which can prevent the decrease in exercising limb blood flow often seen in the elderly (Beere, Russell, Morey, Kitzman, & Higginbotham, 1999; Miyachi, Tanaka, Kawano, Okajima, & Tabata, 2005). Age-related changes in the intrinsic muscle properties, which could impede O2 extraction and utilization in older individuals might also be excluded as factors accounting for the exaggerated development of fatigue characterizing the elderly in the present study. 69 Specifically, healthy aging is associated with a preferential maintenance of fatigue resistant type I oxidative muscle fibers (Lexell, Henriksson-Larsen, Winblad, & Sjostrom, 1983) and capillary contact (Croley et al., 2005) suggesting O2 diffusive properties were likely preserved in the older group (Chilibeck, Paterson, Cunningham, Taylor, & Noble, 1997). Furthermore, although mitochondrial function and O2 utilization typically decline with age (Short et al., 2005), habitually active older participants, such as those in the current study, are not affected by this impairment (Larsen et al., 2012). Finally, aging is associated with a reduced cycling efficiency (Sacchetti, Lenti, Di Palumbo, & De Vito, 2010), increased energetic cost of muscle contraction (Layec et al., 2014), and a greater intramuscular metabolic perturbation during exercise at a given external load (Coggan et al., 1993; Wray et al., 2009). As a result of these age-related changes, older participants accumulate metabolites linked with fatigue (e.g., inorganic phosphate, H+; Allen, Lamb, & Westerblad, 2008; Blain et al., 2016) at a faster rate compared to their younger counterpart (Coggan et al., 1993; Wray et al., 2009). Consequently, this increased rate of accumulation of metabolites likely accounts for a large portion of the observed difference in end-exercise peripheral fatigue between the young and old individuals in this study (Blain et al., 2016). Furthermore, the larger intramuscular metabolic perturbation in older individuals may also contribute to the greater central fatigue which is known to be influenced by the central projection of metabosensitive group III/IV muscle afferents (Sidhu et al., 2017). 70 Development of Fatigue During Exercise at a Given Relative Intensity Time to task failure during both cycling and KE exercise performed at the same relative intensity was similar between young and older participants. However, compared to the old participants, young individuals performed substantially more work during both cycling (+77%) and KE (+33%) exercise. As external work is a key determinant of fatigue (Amann & Dempsey, 2008), this discrepancy might explain the ~10% greater exerciseinduced reduction in MVC presented in the young individuals following both modalities. This greater decrease is largely attributable to a greater degree of central fatigue as peripheral fatigue was not different between groups (Figure 3.3). These data are in agreement with previous studies documenting larger decrements in neuromuscular function in younger individuals following dynamic contractions of the elbow flexors (Yoon, Schlinder-Delap, & Hunter, 2013) and knee extensors (Dalton, Power, Paturel, & Rice, 2015) - despite a similar, or increased, exercise time compared to the older adults. The observation that, despite similar peripheral fatigue and consequently similar metabolic perturbation in both groups, old participants incurred less central fatigue may partly result from the age-related decrease in the central impact of group III/IV muscle afferents (Hoeppli et al., 2016). Specifically, the central projection of metabosensitive muscle afferents in response to a given level of interstitial metabolites is attenuated in older individuals resulting in a lower perception of fatigue compared to their younger counterpart (Hoeppli et al., 2016). Therefore, the comparable peripheral fatigue and metabolic perturbation following exercise at the same relative intensity might have led to a lower central projection of metabosensitive muscle afferents and subsequently less central fatigue in the older participants. However, as central fatigue is determined by a host of factors 71 (Hureau, Romer, & Amann, 2016), other age-related changes might have contributed to the discrepancy in central fatigue following exercise at a given relative intensity. Of note, while seemingly disparate to the discussion of central fatigue within the absolute condition, the greater metabolic perturbation in the older group during an absolute work rate has the potential to offset the attenuated afferent activity with aging. Impact of Aging on the Corticospinal Excitability During Exercise The transmission of neural drive from higher brain areas to contracting muscle occurs predominately through the corticospinal pathway (Brouwer & Ashby, 1992). Alterations in the efficacy of this motor pathway have the potential to influence skeletal muscle activation. Relevant to the current study, aging is associated with a loss of grey and white matter volume (Walhovd et al., 2005) which has the potential to influence corticospinal excitability. Indeed, although not unanimously agreed upon (Clark, Taylor, Hong, Law, & Russ, 2015), aging has been associated with a diminished motor cortical excitability, which is potentially influenced by age-related alterations in intracortical circuitry (Oliviero et al., 2006), and motoneuronal excitability (Rossini, Desiato, & Caramia, 1992). In both exercise modalities and both age groups, the excitability of the corticospinal pathway following fatiguing exercise was unchanged from baseline values, a finding which confirms earlier observations following nonfatiguing exercise (Clark et al., 2015). 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Femoral blood flow (A,C) and leg vascular conductance (B,D) in young and older participants during rhythmic knee extensor exercise performed at the same relative (A,B) and absolute (C,D) exercise intensity. Data are means ± SE. 80 Figure 3.3: Central and peripheral fatigue induced by single-leg knee extensor exercise. A-C show pre- to postexercise values of quadriceps MVC (A), potentiated quadriceps twitch force (B), and voluntary quadriceps activation (VA, C). * P < 0.05, denotes different from Old, # not significantly altered from baseline. Data are means ± SE. 81 Table 3.1: Descriptive characteristics of young and older participants Young Age (yrs) Height (cm) Weight (kg) BMI (kg/m2) Quadriceps muscle mass (kg) MVC (Nm) Qtw (Nm) MRTD (Nm/s) PRR (Nm/s) VA (%) Physical activity Sedentary Light Moderate Vigorous Steps/day Hematologic characteristics RBC (M/ul) Hemoglobin (g/dl) Hematocrit (%) Cholesterol (mg/dl) Triglycerides (mg/dl) HDL (mg/dl) LDL (mg/dl) Old 25 176 75 24 2.0 254 71 1363 524 94 ± ± ± ± ± ± ± ± ± ± 1 2 2 1 0.1 18 5 63 25 2 70 175 79 26 1.9 172 51 972 386 95 1202 141 59 11 8716 ± ± ± ± ± 20 1190 ± 27 20 104 ± 13 10 66 ± 9 5 7 ± 4 391 8932 ± 1227 5.3 15.8 46 160 80 44 101 ± ± ± ± ± ± ± 0.1 0.4 1 6 11 3 6 5.0 15.6 46 179 70 49 109 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1 1 5 2 0.2 10 4 91 46 2 * * * * * 0.1 0.3 1 10 7 2 12 Values are means ± SE. BMI, body mass index; MVC, maximum voluntary contraction of quadriceps; Qtw, potentiated quadriceps twitch; MRTD, maximum rate of torque development; PRR, peak relaxation rate; VA, quadriceps voluntary activation; RBC, red blood cells; HDL, high-density lipoprotein; LDL, low-density lipoprotein. * denotes significant difference between groups, P < 0.05. 82 Table 3.2: Cardiopulmonary and metabolic responses during the final minute of rhythmic knee extension Table 2: Cardiopulmonary and metabolic responses during the final minute of rhythmic knee extension Young Old Young matched Power output (W) KE 54 ± 3 * 45 ± 2 45 ± 2 CYC 249 ± 20 * 155 ± 8 155 ± 8 Exercise time (min) KE 11 ± 1 10 ± 1 10 ± 1 CYC 9 ± 1 8 ± 1 8 ± 1 V̇E (L/min) KE CYC V̇O 2 (L/min) KE CYC V̇O 2 (kg/ml/min) KE CYC V̇CO2 (L/min) KE CYC RER KE CYC V̇E/V̇O 2 KE CYC V̇E/V̇CO 2 KE CYC HR (bpm) KE CYC SV (ml/beat) KE CYC† CO (L/min) KE CYC† MAP (mmHg) KE CYC SVC (ml/min/mmHg) KE CYC RPE KE CYC 84 ± 5 164 ± 7 * 77 ± 7 124 ± 8 41 ± 2 73 ± 5 * * 1.55 ± 0.09 3.68 ± 0.25 * 1.46 ± 0.06 2.38 ± 0.15 1.12 ± 0.07 * 2.51 ± 0.16 20.8 ± 1.1 45.5 ± 2.0 18.3 ± 0.8 29.3 ± 1.8 15.1 ± 0.1 * 31.0 ± 2.5 1.86 ± 0.08 * 4.15 ± 0.28 * 1.57 ± 0.06 2.59 ± 0.17 1.13 ± 0.07 * 2.38 ± 0.16 1.22 ± 0.04 * 1.13 ± 0.03 1.08 ± 0.04 1.10 ± 0.04 1.01 ± 0.03 0.94 ± 0.02 * * 51 ± 3 43 ± 1 * 50 ± 4 51 ± 4 33 ± 1 27 ± 1 * * 42 ± 1 38 ± 1 * 46 ± 3 46 ± 3 33 ± 1 29 ± 1 * 138 ± 7 183 ± 3 * 129 ± 4 150 ± 5 112 ± 4 141 ± 6 77 ± 9 114 ± 6 * 83 ± 12 92 ± 6 91 ± 11 122 ± 6 10.5 ± 1.3 20.3 ± 1.2 10.4 ± 1.2 15.0 ± 0.9 10.0 ± 1.0 17.1 ± 1.0 148 ± 7 147 ± 9 129 ± 5 * * * 72 ± 11 75 ± 12 79 ± 9 10.0 ± 0.0 9.9 ± 0.1 9.9 ± 0.1 9.9 ± 0.1 7.0 ± 0.4 * 5.1 ± 0.9 * Data are means ± SE. KE, single-leg dynamic protocol; CYC, cycling exercise protocol; V̇E, minute ventilation; V̇O2, oxygen consumption; V̇CO2, carbon dioxide production; RER, respiratory exchange ratio; V̇E/V̇O2, ventilatory equivalent for O2; V̇E/V̇CO2, ventilatory equivalent for CO2; HR, heart rate; SV, stroke volume; CO, cardiac output; MAP, mean arterial pressure; SVC, systemic vascular conductance. * denotes significantly different from Old; P < 0.05. CHAPTER 4 HEART FAILURE WITH PRESERVED EJECTION FRACTION IMPAIRS PERIPHERAL HEMODYNAMICS AND RESULTS IN EXAGGERATED NEUROMUSCULAR FATIGUE 84 Abstract This study sought to elucidate the impact of heart failure with preserved ejectionfraction (HFpEF) on neuromuscular fatigue during small muscle mass exercise not limited by centrally-mediated (e.g., cardiac) muscle O2 supply. Eight HFpEF patients (ejectionfraction: 61 ± 2%, NYHA II-III) and healthy-controls (CTRL) performed single-leg kneeextensor exercise to task-failure (80% peak-power [Wpeak]; DYN) and intermittent maximal isometric knee-extensions (8×15-s, 20-s rest, ISO). CTRL also repeated DYN at the same absolute work-rate and duration as HFpEF. Using electrical femoral nerve stimulation, peripheral and central fatigue were quantified via pre/post-exercise changes in quadriceps twitch-torque (Qtw) and voluntary activation (VA). During DYN, exerciseinduced alterations in corticospinal quadriceps excitability were quantified by pre/postexercise changes in motor-evoked potentials. DYN: Exercise time to task-failure at 80% Wpeak (HFpEF: 24 ± 2 W vs. CTRL: 43 ± 2 W; P < 0.05) was comparable (~10 min; P = 0.51) and reduced maximal voluntary quadriceps strength (MVC; -27%), Qtw (-50%), and VA (-6%) equivocally between groups (P > 0.40). Exercise at the same work-rate (24 ± 2 W) and duration (10 ± 1min) reduced MVC (-30 ± 4% vs. -6 ± 4%), Qtw (-54 ± 6% vs. -11 ± 4%), and VA (-6.1 ± 1.4% vs. -0.4 ± 0.7%) significantly more in HFpEF vs. CTRL. Corticospinal excitability remained unaltered during exercise in both groups (P > 0.15). ISO: Although quadriceps MVC was similar between groups at baseline (~160 Nm), HFpEF demonstrated a greater (P < 0.05) exercise-induced reduction in MVC (-26 ± 4% vs. -13 ± 4%) and Qtw (-49 ± 4% vs. -23 ± 7%). In conclusion, HFpEF exacerbates the development of both central and peripheral fatigue. Therefore, this impairment may primarily be determined by disease-related alterations in the periphery. 85 Introduction Although heart failure has historically been associated with a weakened heart and a reduced ejection fraction (HFrEF), it is now recognized that approximately one-half of all heart failure patients characterized by a preserved ejection fraction (≥50%; HFpEF) (Owan et al., 2006). Despite significant pathophysiological differences (for review see Reddy & Borlaug, 2016), patients with HFpEF exhibit a similar morbidity and mortality as those with HFrEF (Bhatia et al., 2006; Owan et al., 2006) and suffer from severe exercise intolerance (Borlaug et al., 2006; Kitzman et al., 2014; Parthasarathy et al., 2009). However, the underlying mechanisms of this impairment are not fully understood. In addition to distinct central, that is, cardiac, abnormalities (Borlaug, Lam, Roger, Rodeheffer, & Redfield, 2009; Borlaug et al., 2006; Haykowsky et al., 2011; Phan et al., 2010), recent studies revealed significant impairments in numerous peripheral mechanisms determining convective oxygen transport and its utilization by the skeletal muscle in HFpEF (Bhella et al., 2011; Dhakal et al., 2015; Kitzman et al., 2002; Kitzman et al., 2014; Lee et al., 2016). As oxygen supply and utilization are key determinants of fatigue (Amann & Calbet, 2008), these peripheral impairments could exacerbate the development of neuromuscular fatigue during physical activities and thereby explain, at least in part, the severe exercise intolerance characterizing patients with HFpEF (Dhakal et al., 2015; Haykowsky et al., 2011). However, there is currently no direct evidence for an altered development of neuromuscular fatigue during physical activities in these patients. Exercise-induced neuromuscular fatigue is defined as a reversible decrease in the torque or power generating capacity of a muscle or muscle group (Gandevia, 2001). This temporary impairment can result from neuronal changes within the central nervous system 86 causing a decrease in neural activation of the muscle (i.e., central fatigue) and/or biochemical changes at or distal to the neuromuscular junction causing an attenuated contractile response to neural input (i.e.,, peripheral fatigue ; Bigland-Ritchie, Jones, Hosking, & Edwards, 1978; Taylor, Amann, Duchateau, Meeusen, & Rice, 2016). Peripheral fatigue can be examined by quantifying the exercise-induced fall in muscle twitch torque (Merton, 1954). Central fatigue can be evaluated by quantifying the exerciseinduced fall in a participant's ability to voluntarily activate a muscle (VA; Gandevia, 2001). Importantly, as VA is critically dependent on descending neural input reaching skeletal muscle, the integrity and excitability of the motor pathway linking the brain with the exercising muscle, termed the corticospinal pathway, is important and has been considered a potential contributor to central fatigue (Martin, Smith, Butler, Gandevia, & Taylor, 2006; Petersen, Taylor, Butler, & Gandevia, 2003). Alterations in the excitability of the corticospinal pathway can be assessed with transcranial magnetic stimulation (TMS; Barker, Jalinous, & Freeston, 1985). Specifically, this noninvasive method evokes short latency electromyographic (EMG) responses, termed motor-evoked potentials (MEP), in a target muscle. When normalized to the maximal compound muscle action potential (Mmax), activity-induced alterations in MEP size reflect changes in the excitability of corticospinal pathway (Day et al., 1989; Rothwell, Thompson, Day, Boyd, & Marsden, 1991) It was the goal of this investigation to evaluate the impact of HFpEF on the development of neuromuscular fatigue during single-leg knee extensions performed both at the same relative and absolute exercise intensity. Single-joint exercise was chosen over whole body exercise as it minimizes the confounding influence of the patients' malfunctioning cardiac pump (Esposito, Mathieu-Costello, Shabetai, Wagner, & 87 Richardson, 2010). It was hypothesized that HFpEF patients would develop more neuromuscular fatigue during exercise compared to their healthy counterparts (CTRL). Methods Participants Eight patients with echocardiographic evidence for HFpEF (6 males, 2 females) and 8 sex- and age-matched CTRLs participated in this study. Descriptive characteristics are presented in Table 4.1. Echocardiographic and medication data for the HFpEF participants are presented in Table 4.2. Written informed consent was obtained from all participants before their inclusion in this study. The Institutional Review Boards of the University of Utah and the Salt Lake City Veterans Affairs Medical Center approved all protocols. Experimental Procedures During 3 preliminary visits, anthropometric measurements were collected and participants were familiarized with exercise and procedures of both experimental protocols. All exercise sessions were separated by at least 24 hours and performed on the dominant leg (right). Quadriceps fatigue was quantified by the pre- to postexercise decrease in neuromuscular function. Exercise-induced alterations in corticospinal excitability were quantified by the pre- to postexercise change in motor-evoked potentials (MEP) normalized to Mmax. 88 Protocol A - Rhythmic submaximal single-leg knee extension exercise: The experimental protocol is depicted in Figure 4.1 A. Prior to exercise and the assessment of baseline neuromuscular function, participants performed a 1-minute unloaded warm-up on a custom-made knee extensor ergometer. The first preliminary visit consisted of a maximal incremental single-leg knee extensor exercise test. Subjects were asked to maintain a consistent cadence at 60 rpm throughout all testing procedures. Peak power output (Wpeak) and oxygen consumption (VO2peak) were determined by increasing the work rate from an unloaded state by 5-10 W·min-1 until task failure (rpm drops below 50 for >5 s despite verbal encouragement). Wpeak represents the workload during the last stage completed while VO2peak represents the average O2 consumption during the last minute of exercise. During the 2nd and 3rd visit, participants performed familiarization trials at 80% Wpeak to task failure (Tlim). During the 4th visit, considered the experimental day, participants performed the 80% Tlim exercise and exercise-induced neuromuscular quadriceps fatigue and changes in corticospinal excitability were assessed. The CTRLs returned for a 5th visit in which they matched HFpEF on a person-by-person basis (i.e., the CTRL with the highest Wpeak matched the HFpEF with the highest Wpeak). Protocol B - Isometric maximal single-leg knee extensions: The experimental protocol is depicted in Figure 4.1 B. Prior to exercise and the assessment of baseline neuromuscular function, participants performed a standardized warm-up consisting of ten 5-s contractions equaling 20% of the participant's maximal voluntary quadriceps contraction (MVC) strength. Afterwards, participants performed eight intermittent maximal contractions (15-s MVC each followed by a 20-s rest period). Femoral nerve stimulation (FNS) was performed at the end of each MVC and 2 s into the rest phase to 89 quantify potentiated quadriceps twitch torque (Qtw) and calculate VA during the protocol (Figure 4.1 B). General Instrumentation Physical activity level and 6-minute walk test: Once instructed on proper operating procedures, all participants wore an accelerometer (GTIM; Actigraph, Pensacola, FL) for ten consecutive days after completing the study. Total daily physical activity was averaged and reported as steps per day and physical activity counts per minute. The counts per minute were subsequently categorized into sedentary, low, moderate, and vigorous intensities via commercially available software (Actilife, Actigraph, Pensacola, FL). Additionally, during the second preliminary visit, HFpEF patients performed a 6-minute walk test (Cahalin, Mathier, Semigran, Dec, & DiSalvo, 1996). Cardiovascular measures: Heart rate was determined via the R-R interval obtained from a 12-lead electrocardiogram acquired with a data acquisition system (Spike2, Cambridge Electronic Design Ltd, Cambridgeshire, England, UK). Mean arterial pressure (MAP) was determined via a Finometer placed at heart level utilizing finger photoplethsmography (Finapres Medical Systems, Amsterdam, The Netherlands). Stroke volume (SV) was estimated via beat-by-beat assessment of the pressure waveform using the Modelflow method (Beatscope, version 1.1, Finapress Medical Systems). Cardiac output was calculated as the product of HR and SV. Systemic vascular conductance (SVC) was calculated as the quotient of cardiac output and MAP. Electromyography: The EMG signals were obtained using surface electrodes (Ag- 90 AgCl, 10-mm diameter) placed on the muscle belly and tendon of the vastus lateralis (VL) in a monopolar configuration. Prior to electrode placement, the skin was lightly abraded with fine sandpaper and cleaned with an alcohol swab. EMG signals were amplified 1000 times (Neurolog Systems, Digitimer Ltd., Welwyn Garden City, Hertfordshire, England, UK), band-pass filtered (20-1000 Hz; NL-844, Digitimer Ltd), and converted from analog to digital at a sampling rate of 2000 Hz using a 16-bit Micro 1401 mk-II and Spike2 data collection software (Cambridge Electronic Design Ltd, Cambridgeshire, England, UK) running custom written scripts. During the rhythmic knee extensor exercise, knee angle was monitored continuously. The knee angle at which the largest VL EMG burst occurs was identified as the centering point for EMG averaging (100 ms including 50 ms before and 50 ms after the centering point [Weavil, Sidhu, Mangum, Richardson, & Amann, 2015]) and measured throughout exercise. FNS: The EMG signals were obtained using surface electrodes (Ag-AgCl, 10-mm diameter) placed on the muscle belly and tendon of the vastus lateralis (VL) in a monopolar configuration. Prior to electrode placement, the skin was lightly abraded with fine sandpaper and cleaned with an alcohol swab. EMG signals were amplified 1000 times (Neurolog Systems, Digitimer Ltd., Welwyn Garden City, Hertfordshire, England, UK), band-pass filtered (20-1000 Hz; NL-844, Digitimer Ltd), and converted from analog to digital at a sampling rate of 2000 Hz using a 16-bit Micro 1401 mk-II and Spike2 data collection software (Cambridge Electronic Design Ltd, Cambridgeshire, England, UK) running custom written scripts. During the rhythmic knee extensor exercise, knee angle was monitored continuously. The knee angle at which the largest VL EMG burst occurs was identified as the centering point for EMG averaging (100 ms including 50 ms before 91 and 50 ms after the centering point [Weavil et al., 2015]) and measured throughout exercise. Neuromuscular quadriceps function and torque acquisition: To examine exerciseinduced quadriceps fatigue, neuromuscular knee extensor function of the exercising leg was assessed before and immediately after exercise (post-exercise measures occurred 37 ± 2 s after rhythmic exercise and 9 ± 1 s after isometric exercise). Participants were seated upright in the knee extensor ergometer with their hip and knee flexed at 120° and 90°, respectively. A noncompliant cuff placed 2-3 cm superior to the right lateral malleolus was attached to a calibrated load cell (MLP 300; Transducer Techniques, Inc. Temecula, CA, USA). Cuff position remained secured throughout each session. The assessment of quadriceps function included three MVCs, over which a FNS was elicited. If muscular activation was suboptimal during the MVC, the FNS would evoked a superimposed twitch (SIT). Following each MVC, another FNS stimulation was initiated to evoke a resting potentiated quadriceps twitch (Qtw). Voluntary quadriceps activation (VA) was calculated as: VA = [1-(SIT/Qtw)]*100 (Belanger & McComas, 1981; Merton, 1954). Exerciseinduced peripheral and central fatigue was quantified as the percent reduction of the averaged Qtw and VA, respectively, from before to immediately after exercise. Furthermore, the maximal rate of torque development (MRTD, calculated as the highest positive derivative of the torque during a 10-ms interval) and peak relaxation rate (PRR, the highest negative derivative of the torque during a 10-ms interval) were analyzed for each Qtw. 92 Protocol Specific Instrumentation Protocol A - Rhythmic submaximal single-leg knee extension exercise: Ventilation, pulmonary gas exchange and cardiovascular measures: Ventilation and pulmonary gas exchange were measured at rest and during exercise with a metabolic cart (Innocor, Innovision, Odense, Denmark). Participant's rating of perceived exertion (RPE) was obtained at the end of each minute during exercise using Borg's Category Ratio 10 scale (Borg, 1982). Leg blood flow: Femoral blood flow measurements were measured at rest and during exercise for visits 1 (max test) and 4 (Tlim exercise) as previously described (Groot et al., 2013; Wray & Richardson, 2006). Briefly, a Logic 7 ultrasound (General Electric Medical Systems, Milwaukee, WI) was used for measurements of vessel diameter and blood velocity of the common femoral artery (CFA), proximal to the bifurcation of the superficial and deep femoral artery. Using CFA diameter and mean blood velocity (Vmean), femoral blood flow (mL·min-1) was calculated as: Vmean·π·(vessel diameter/2)2·60. The ultrasound probe was positioned to maintain an insonation angle of 60° or less during all blood velocity measurements. Leg vascular conductance was calculated as femoral blood flow /MAP. EMG analysis: Knee angle was monitored throughout exercise. During a brief (~45 s) bout of exercise at the experimental workload, the knee angle in which the peak EMG burst of the VL (i.e., the centering point, knee angle: 89 ± 1°) was determined. For EMG analysis during exercise, 10 seconds of rectified EMG waveforms were overlaid and averaged around the centering point EMG was normalized to that obtained during the first minute of exercise. 93 TMS: To begin, stimuli were delivered over the vertex of the motor cortex (left hemisphere, approximately 2-3 cm lateral of the vertex) using a concave double-cone coil (Magstim 200; Magstim Co. Ltd, Whitland, UK) to elicit MEPs in the right quadriceps during all protocols. Optimal positioning of the TMS coil was determined prior to the experiment and marked on the scalp for accurate placement throughout the study. To ensure that the MEP was submaximal at the beginning of exercise, thereby allowing room for measureable alterations, TMS stimulator intensity was set at 120% of the resting motor threshold (i.e., the stimulation intensity which evokes a MEP in at least 4 of 7 stimulations) (CTRL: 53 ± 4%; HFpEF: 57 ± 8%; P = 0.86). To quantify exercise-induced changes in corticospinal excitability, participants received three stimulation sets before and again after exercise, consisting of three TMS and one FNS. These stimulations were randomized during a constant quadriceps contraction equating to 20% of the EMG during a preexercise MVC (Sidhu et al., 2017; Figure 4.1 A). Data Analysis All data were stored and analyzed offline using Spike2 data acquisition software. The area for each MEP and Mmax was measured and averaged over the three stimulation sets. To account for potential changes within the muscle, MEPs were normalized to Mmax. Statistical Analysis A priori comparisons were performed between the HFpEF and control groups for both the relative and absolute conditions. Two-way mixed model ANOVAs (group by workload and group by time) were performed for the comparison of LBF during the 94 rhythmic knee extension exercise max test and the Tlim trial, respectively. Additionally, a two-way mixed model ANOVA was performed for alterations in neuromuscular function (MVC, Qtw, and VA) during the isometric trial. The area under the curve (AUC) was analyzed for the comparison of cardiovascular changes during the isometric trial. Student's t tests were employed to compare descriptive characteristics, pre- to postexercise changes in neuromuscular function (MVC, Qtw, VA, MRTD, PRR, and Mmax), corticospinal excitability (normalized MEP), and end-exercise metabolic and ventilatory data. Alpha was set at 0.05. All data is reported as mean ± standard error of the mean (SE). Results Baseline Neuromuscular Function Preexercise MVC, Qtw, and VA were similar between CTRL and HFpEF participants (P > 0.38) (Table 4.1). Furthermore, MRTD (HFpEF: 904 ± 102 Nm/s, CTRL: 996 ± 74 Nm/s; P = 0.69) and PRR (HFpEF: 356 ± 51 Nm/s, CTRL: 403 ± 44 Nm/s; P = 0.66) were equivocal between groups. Protocol A: Rhythmic Submaximal Knee Extension Exercise Incremental exercise test: Wpeak was nearly 75% higher in CTRL compared to HFpEF (P < 0.01, Table 4.1). At baseline, femoral blood flow (296 ± 26 ml/min vs. 262 ± 35 ml/min, P = 0.44) and leg vascular conductance (3.4 ± 0.4 ml/min/mmHg v 2.9 ± 0.3 ml/min/mmHg; P = 0.34) were similar between CTRLs and HFpEF, respectively. However, femoral blood flow and leg vascular conductance were lower at all workloads in 95 HFpEF compared to CTRL (group, F1,13 > 11.4, P <0.05, time, F1,13 > 16.6, P < 0.001). Specifically, CTRLs demonstrated a significantly greater femoral blood flow at 0 W (1408 ± 81 ml/min vs. 1061 ± 105 ml/min), 10 W (1752 ± 72 ml/min vs. 1299 ± 146 ml/min), 20 W (2166 ± 100 ml/min vs. 1560 ± 172 ml/min) and peak exercise (3387 ± 210 ml/min vs. 1975 ± 194 ml/min, P < 0.05). Additionally, leg vascular conductance was greater in CTRLs at 10 W (15.8 ± 0.7 ml/min/mmHg vs. 11.6 ± 1.2 ml/min/mmHg, P < 0.05), 20 W (19.0 ± 0.8 ml/min/mmHg vs. 13.1 ± 1.4 ml/min/mmHg, P < 0.05), and at peak exercise (22.9 ± 1.2 ml/min/mmHg vs. 14.6 ± 1.8 ml/min/mmHg, P < 0.05). VE (62.1 ± 45 L/min vs. 41.1 ± 6.1 L/min), VO2 (1.18 ± 0.06 L/min vs. 0.90 ± 0.07 L/min), VCO2 (1.50 ± 0.09 vs. 1.00 ± 0.05 L/min), and RER (1.29 ± 0.07 vs. 1.09 ± 0.05) were all significantly greater at end exercise in the CTRLs compared to HFpEF. Exercise at the same relative intensity and to task failure: Calculated coefficients of variation for the time to task failure were 8% and 12% in CTRL and HFpEF participants, respectively. While the CTRL group completed more total work during the Tlim trial (29 ± 4 kJ vs. 15 ± 3 kJ, P < 0.05), endurance time to task failure was similar (CTRL: 668 ± 70 s vs. HFpEF: 606 ± 74 s; P = 0.55) between groups. VL EMG progressively increased to task failure, however the increase in EMG (~100%, P = 0.93) was not different between CTRL and HFpEF. Femoral blood flow (group, F1,13 = 28.4, P < 0.001; time, F1,13 = 17.3, P < 0.05) and leg vascular conductance (group, F1,13 = 4.85, P < 0.05; time, F1,13 = 3.90, P < 0.05) were greater at each time point during the Tlim exercise in CTRL compared to HFpEF (Figure 4.2). Additional cardiovascular and metabolic data are shown in Table 4.3. The exercise-induced changes in neuromuscular function are reflected in Figure 4.3. Considering the amount of work performed, HFpEF demonstrated a ~90% greater 96 impairment in Qtw per unit of work compared to CTRLs (1.63 ± 0.36 Nm/kJ vs. 0.86 ± 0.18 Nm/kJ; P < 0.05). Additionally, MRTD (-67 ± 4% vs. -47 ± 6%; P < 0.05) and PRR (-61 ± 5% vs. -37 ± 4%; P < 0.05) were diminished to a greater extent in the HFpEF group. Pre- and postexercise Mmax were similar in both HFpEF (29.2 ± 9.1 μV·s vs. 29.9 ± 9.0 μV·s, P = 0.31) and CTRLs (30.7 ± 5.6 μV·s vs. 30.8 ± 5.4 μV·s, P = 0.82). MEPs (normalized to Mmax) remained unchanged from pre- to postexercise in HFpEF (31± 6% vs. 29.6 ± 5.0%, P = 0.15) and CTRLs (34.5 ± 4.6% vs. 33.3 ± 4.7%, P = 0.72). Exercise at the same absolute intensity and for the same duration: By design, knee extensor exercise time and power output were matched between groups. VL EMG remained unaltered from the first minute of exercise to the end of exercise in CTRLs (P = 0.69) while it increased by 95 ± 26% (P < 0.05) during exercise in HFpEF. Femoral blood flow was ~75% higher in CTRL compared to HFpEF (time, F1,13 = 19.06, P <0.001; group, F1,13 = 7.2, P < 0.05), despite having a similar cardiac output (Figure 4.2). Furthermore, leg vascular conductance was ~80% higher in CTRL compared to HFpEF (time, F1,13 = 10.35, P < 0.05; group, F1,13 = 7.82, P < 0.05; Figure 4.2). Additional cardiovascular and metabolic responses are shown in Table 4.3. The exercise-induced changes in neuromuscular function are reflected in Figure 4.3. Additionally, the change in Qtw per unit of work was greater in the HFpEF compared to CTRL (1.63 ± 0.36 Nm/kJ vs. 0.36 ± 0.14 Nm/kJ, respectively; P < 0.05). The greater degree of peripheral fatigue in HFpEF was also reflected in the pre- to post-exercise change in within twitch variables as MRTD (-12 ± 4%, P < 0.05) and PRR (-8 ± 5 %, P <0.05) declined to a smaller extent in CTRL. Pre- and postexercise Mmax were similar in both HFpEF (29.2 ± 9.1 μV·s vs. 29.9 ± 9.0 μV·s, P = 0.31) and CTRL (37.0 ± 2.9 μV·s vs. 36.8 ± 3.2 μV·s, P = 0.65). 97 Additionally, normalized MEPs (normalized to Mmax) remained unchanged from pre- to post-exercise in HFpEF (31± 6% vs. 29.6 ± 5.0 %, P = 0.15) and CTRL (35.0 ± 5.4% vs. 34.9 ± 7.2 mV, P = 0.95). Protocol B: Maximal Isometric Knee Extensions Baseline HR (73 ± 1 bpm vs. 82 ± 5 bpm, P = 0.12), SV (74 ± 10 ml/beat vs. 73 ± 6 ml/beat, P = 0.91), CO (5 ± 1 L/min vs. 6 ± 1 L/min, P = 0.26), MAP (95 ± 7 mmHg vs. 102 ± 5 mmHg, P = 0.64), and SVC (52 ± 6 ml/min/mmHg vs. 65 ± 9 ml/min/mmHg, P = 0.31) were similar for CTRLs and HFpEF, respectively. The cardiovascular responses to the isometric protocol are illustrated in Figure 4.4. MVC torque at the beginning of exercise was similar in both groups and progressively declined throughout the protocol. There was a significant interaction for MVC (interaction, F1,13 = 3.25, P < 0.05) and a greater fall in HFpEF compared to CTRL (Figure 4.5). Furthermore, Qtw was similar between groups at the beginning of exercise but fell to a greater extent in the HFpEF compared to CTRL (interaction, F1,13 = 2.59, P < 0.05) (Figure 4.5). In contrast, VA remained unchanged throughout the protocol in both groups (group, F1,13 = 1.73, P = 0.21; time, F1,13 = 0.65, P = 0.72; interaction, F1,13 = 1.29, P = 0.27) (Figure 4.5). Finally, MRTD (-53 ± 6% vs. -27 ± 6%; P < 0.01) and PRR (-51 ± 6% vs. 26 ± 7%; P < 0.05) were impaired to a greater extent in HFpEF compared to the CTRL. 98 Discussion The current study sought to quantify the impact of HFpEF on the development of neuromuscular fatigue during exercise involving a small muscle mass. The main finding was that HFpEF is associated with an exacerbation of both central and peripheral fatigue during physical activity minimally influenced by the patients' malfunctioning cardiac pump. Given the role of neuromuscular fatigue as a limiting factor of exercise, this diseaserelated impairment likely accounts for a significant portion of the exercise intolerance characterizing patients with HFpEF. Submaximal Rhythmic Knee Extension Exercise Time to task failure during rhythmic KE exercise performed at the same relative intensity (80% Wpeak) was similar in HFpEF and CTRLs and caused a comparable degree of central and peripheral fatigue (Figure 4.3). These similarities occurred despite the substantially lower power output during exercise in HFpEF (24 W vs. 46 W) and therefore indirectly suggest a greater susceptibility to fatigue in the patients. Indeed, HFpEF patients demonstrated a nearly 90% greater decline in peripheral neuromuscular function per unit of work. Additional experiments further highlighted this impairment in HFpEF and revealed, compared to CTRL, a 3-4 fold greater degree of central and peripheral fatigue following exercise performed at the same work load (24 W) and for the same duration (10 min). As fatigue is considered a major factor limiting physical activity (Amann & Dempsey, 2008; Amann et al., 2006; Hureau, Olivier, Millet, Meste, & Blain, 2014), the previously documented exercise intolerance in HFpEF (Borlaug et al., 2006; Kitzman et 99 al., 2014; Parthasarathy et al., 2009) may therefore, at least partially, be accounted for by the patients' impaired neuromuscular fatigue resistance. Although the present findings in HFpEF appear comparable to observations in HFrEF (Hopkinson et al., 2013), appropriately designed studies are needed to evaluate exact differences and similarities between the two etiologies. Oxygen supply to skeletal muscle is a significant determinant of endurance capacity and the development of peripheral fatigue during physical activity (Amann & Calbet, 2008) and is limited by central (i.e., cardiac output) and peripheral (i.e., vascular conductance) factors (Saltin, 1988). As this study aimed to emphasize the impact of peripheral abnormalities on neuromuscular fatigue, rhythmic KE exercise, which is, even in heart failure, not compromised by cardiac output (Andersen & Saltin, 1985; Esposito et al., 2010), was chosen as the exercise modality. Indeed, owing to the patients' elevated chronotropic response, cardiac output during exercise was similar in HFpEF and CTRLs (Table 4.3). However, femoral blood flow and leg vascular conductance were significantly lower during exercise at a given work rate in HFpEF compared to CTRLs (Figure 4.2; Lee et al., 2016). Given the similar [Hb] (Table 4.1), and likely a comparable oxygen saturation during exercise in both groups (Amann et al., 2014; Esposito et al., 2010), this observation suggests peripheral O2 supply limitation as a key factor compromising the patients' fatigue resistance. Although only little is currently known about the underlying mechanisms of these hemodynamic abnormalities (Wray, Amann, & Richardson, 2017), an exaggerated exercise pressor reflex may partly account for this impairment (Amann et al., 2014). In addition to peripherally mediated O2 supply limitations, the exaggerated peripheral fatigue in HFpEF is likely also determined by impaired O2 extraction (Bhella et 100 al., 2011; Dhakal et al., 2015), utilization (Kitzman et al., 2002), and a disease-related shift from fatigue resistant oxidative towards fatigue-prone glycolytic muscle fibers (Kitzman et al., 2014). Taken together, impaired peripheral mechanisms of O2 transport and utilization combined with a shift in intrinsic muscle characteristics likely facilitate the intramuscular accumulation of fatigue metabolites (Allen, Lamb, & Westerblad, 2008; Blain et al., 2016) in HFpEF and thereby account for a large part of the patients' increased susceptibility to peripheral fatigue. The development of central fatigue during exercise was also significantly exaggerated in HFpEF compared to CTRL (Figure 4.3). Although the disease might have an impact on various central fatigue related processes within the central nervous system, neural feedback mechanisms from the exercising quadriceps presumably played a key role in this discrepancy. Specifically, group III/IV muscle afferents, which respond to mechanical stress and metabolic perturbations within exercising muscle (Adreani, Hill, & Kaufman, 1997; Kaufman & Rybicki, 1987), project to various sites within the central nervous system (Craig, Bushnell, Zhang, & Blomqvist, 1994; Liu et al., 2003; Sidhu et al., 2017) and facilitate the development of central fatigue during physical activities (Amann, Proctor, Sebranek, Pegelow, & Dempsey, 2009; Sidhu et al., 2017; Taylor et al., 2016). Given the tight relationship between intramuscular metabolic perturbations and peripheral fatigue (Blain et al., 2016) and the greater peripheral fatigue in HFpEF, group III/IV muscle afferent feedback was likely higher in the patients compared to CTRL and may account for their substantially larger degree of end-exercise central fatigue (Sidhu et al., 2017). Although the exact relevance of corticospinal excitability as a contributor to central fatigue remains to be determined (Gandevia, 2001; Klass, Levenez, Enoka, & Duchateau, 101 2008; Martin, Gandevia, & Taylor, 2006; Sidhu et al., 2017; Taylor, Petersen, Butler, & Gandevia, 2000), the current study investigated the impact of HFpEF on exercise-induced changes in the efficacy of the central motor pathway to relay neural signals. Specifically, alterations in corticospinal excitability can influence the amount of neural input from higher brain areas to skeletal muscle and therefore alter voluntary movements and muscle activation (Martin, Smith, et al., 2006; Petersen et al., 2003). However, since prolonged exercise had no effect on the net excitability of the corticospinal pathway in HFpEF or CTRL, this mechanism might be excluded as a potential determinant of the patients' greater degree of end-exercise central fatigue. Maximal Intermittent Isometric Knee Extensions As the development of fatigue during exercise is highly task specific (Enoka, 1995), the current study evaluated whether the patients' compromised fatigue resistance observed during the rhythmic exercise is also reflected during a different small muscle mass exercise modality, that is, maximal, intermittent, isometric quadriceps contractions. Despite starting at similar baseline values, quadriceps MVC fell to a greater extent in HFpEF compared CTRLs and this discrepancy mainly resulted from the exaggerated development of peripheral fatigue (Figure 4.4). Although these findings during the isometric protocol generally corroborate the impaired fatigue resistance of HFpEF during the rhythmic exercise, it could be argued that the patients' compromised cardiac pump might have influenced this observation. Specifically, while cardiac output increased from rest during this protocol in CTRL, it remained unaltered in HFpEF. Given the known sensitivity to left 102 ventricular afterload in heart failure (Kitzman, Higginbotham, Cobb, Sheikh, & Sullivan, 1991; Melenovsky, Hwang, Lin, Redfield, & Borlaug, 2014), the lack of an increase in cardiac output was likely secondary to the substantial fall in systemic vascular conductance (which remained unaltered in CTRL) during the exercise. The isometric protocol might therefore not be a suitable exercise modality for investigating the role of the periphery in the development of fatigue during small muscle mass exercise in heart failure. Experimental Considerations Typical of this patient population, the HFpEF participants in this study exhibited various comorbidities such as coronary artery disease, diabetes, and hypertension. These patients did not abstain from taking prescribed medications and thus were studied in their optimally-medicated state. As such, we cannot exclude a possible pharmacotherapy effect on the reported cardiovascular responses and/or fatigue measures. Although this may introduce heterogeneity in terms of the response to exercise, it allows for the study of HFpEF in a manner which represents the diverse nature of this population. Finally, the present findings are limited to single-joint exercise. Specifically, the greater strain on the heart during whole body exercise may cause an even greater discrepancy in the development of neuromuscular fatigue between HFpEF and CTRL. Patients with HFpEF are characterized by an exacerbated development of central and peripheral fatigue during exercise involving a small muscle mass. This impairment is largely attributable to disease-related peripheral abnormalities including skeletal muscle oxygen supply, utilization, and likely intrinsic muscle characteristics. Since neuromuscular 103 fatigue plays a key role in determining exercise intolerance and functional capacity, pharmaceutical treatment strategies and physical rehabilitation programs may benefit from targeting these peripheral abnormalities in HFpEF. References Adreani, C. M., Hill, J. M., & Kaufman, M. P. (1997). Responses of group iii and iv muscle afferents to dynamic exercise. Journal of Applied Physiology, 82(6), 1811-1817. Allen, D. G., Lamb, G. D., & Westerblad, H. (2008). 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W., & Richardson, R. S. (2006). Aging, exercise, and limb vascular heterogeneity in humans. Medicine and Science in Sports and Exercise, 38(10), 1804-1810. doi: 10.1249/01.mss.0000230342.86870.94 109 Figure 4.1: Schematic illustration of the exercise protocol. Protocol A included the assessment of neuromuscular and corticospinal function before- and after-exercise. Fatigue during exercise in protocol B was quantified using femoral nerve stimulations (F). T, transcranial magnetic stimulation; Tlim, time to task failure; 20% EMG contraction, 20% of the pre-exercise MVC EMG. 110 Figure 4.2: Hemodynamic response to rhythmic knee extensor exercise. Femoral blood flow (A,D), mean arterial pressure (B,E) and leg vascular conductance (C,F) in control participants (CTRL) and patients with HFpEF during rhythmic knee extensor exercise at the same relative (A,B,C) and absolute (D,E,F) exercise intensity. Data are means ± SE. 111 Figure 4.3: Central and peripheral fatigue induced by rhythmic knee extensor exercise. Exercise was performed at the same relative (left) and absolute (right) exercise intensity. Data are means ± SE.* denotes significant difference between groups, # not significantly different from baseline, P < 0.05. 112 Figure 4.4: Second-by-second plots of cardiovascular responses during isometric exercise. Heart rate (A), stroke volume (B), cardiac output (C), mean arterial pressure (D), and systemic vascular conductance (E) for healthy controls (open) and heart failure with preserved ejection fraction (HFpEF, closed). Black horizontal bars represent maximal 15 s isometric contractions. Statistical analysis was performed on the area under the curve, data are means ± SE. * denotes statistical significance, P ≤ 0.05. 113 Figure 4.5: Central and peripheral fatigue during intermittent isometric exercise. Data are means ± SE. * denotes significant difference between groups, P < 0.05. 114 Table 4.1: Descriptive characteristics of healthy controls and HFpEF CTRL 67 ± 1 174 ± 1 76 ± 5 25 ± 2 53 ± 2 159 ± 7 45 ± 4 96 ± 2 Age (yrs) Height (cm) Weight (kg) BMI (kg/m 2 ) KEmax (W) MVC (Nm) Qtw (Nm) VA (%) Physical activity Sedentary (min/day) 1196 ± Light (min/day) 102 ± Moderate (min/day) 64 ± Vigorous (min/day) 7± Steps/day (counts/day) 7597 ± Hematologic characteristics RBC (M/ul) 4.7 ± Hemoglobin (g/dl) 14.9 ± Hematocrit (%) 44 ± Glucose (mg/dl) 86 ± Cholesterol (mg/dl) 186 ± Triglycerides (mg/dl) 74 ± HDL (mg/dl) 60 ± LDL (mg/dl) 115 ± HFpEF 64 ± 1 173 ± 2 114 ± 10 * 38 ± 3 * 31 ± 3 * 149 ± 15 43 ± 5 94 ± 2 29 14 9 4 697 1273 90 16 0 3688 ± ± ± ± ± 37 23 5 * 0 711 * 0.1 0.2 1 4 7 3 5 11 5.0 14.8 45 135 165 229 40 85 ± ± ± ± ± ± ± ± 1.0 1.0 2 19 22 47 * 4 * 17 Values are means ± SE. BMI, body mass index; KEmax, peak workload attained during incremental KE exercise; MVC, maximum voluntary contraction of quadriceps; Qtw, potentiated quadriceps twitch; VA, quadriceps voluntary activation; RBC, red blood cells; HDL, high-density lipoprotein; LDL, low-density lipoprotein. * denotes significant difference between groups, P < 0.05. 115 Table 4.2: Medications and echocardiography based characteristics of HFpEF Disease related characteristics NYHA Class II III Atrial fibrillation CAD COPD Diabetes Hypertension Six min walk distance (m) B-Type natriuretic peptide (pg/ml) Echocardiography Ejection fraction (%) LV IVSd (cm) LV PWd (cm) LV ID diastole (cm) LV ID systole (cm) LA ESV index (ml/m 2 ) TR gradient (mmHg) Peak E wave (cm/s) Peak A wave (cm/s) E/A ratio E' lateral wall (cm/s) E' septal wall (cm/s) E/E' lateral E/E' septal E/E' ratio (average) Mitral E-wave deceleration (ms) Peak TR velocity (m/s) Medications Beta receptor blockers ACEi or ARB Loop diuretics Statin Nitrates Calcium channel blockers 4 (50%) 4 (50%) 2 (25%) 3 (38%) 1 (13%) 2 (25%) 4 (50%) 387 ± 43 132 ± 55 Normal Reference 61 1.1 1.1 4.8 3.2 39 44 95 72 1.6 8 6 12 17 14 236 3.1 4 5 4 2 2 5 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 2 0.1 0.0 0.2 0.2 4 3 7 10 0.3 0 1 1 2 1 21 0.1 ≥ 55 0.6 - 1.1 0.6 - 0.9 3.9 - 5.3 2.0 - 4.0 < 34 ≤ 50 ≤ 0.8 > 10 >7 < 13 < 15 < 14 < 2.8 (50%) (63%) (50%) (25%) (25%) (63%) Values are means ± SE or % of group. HFpEF, heart failure with preserved ejection fraction; NYHA, New York Heart Association; CAD, coronary artery disease; COPD, chronic obstructive pulmonary disease; LV IVSD, left ventrical interventricular septum diameter; LV PWD, left ventricular posterior wall diameter; LV ID, left ventricular internal diameter; LA ESV, left atrium end systolic volume; TR, tricuspid regurgitation; ACEi, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker. 116 Table 4.3: Cardiopulmonary and metabolic responses during the final minute of rhythmic knee extension performed at the same relative and absolute intensity * HFpEF 24 ± 2 Control matched 24 ± 2 * 10.1 ± 0.5 99 ± 7 10.1 ± 0.5 85 ± 3 Power output (W) Control 43 ± 2 Exercise time (min) HR (bpm) 11.2 ± 1.2 130 ± 5 SV (ml/beat) CO (L/min) 84 ± 11 10.6 ± 1.1 108 ± 15 10.2 ± 1.2 111 ± 7 8.8 ± 0.5 MAP (mmHg) V̇E (L/min) V̇O2 (L/min) V̇O2 (kg/ml/min) 146 ± 9 75 ± 7 139 ± 4 48 ± 5 115 ± 7 35 ± 2 1.41 ± 0.07 * 18.2 ± 0.8 * 1.00 ± 0.07 9.2 ± 1.1 0.79 ± 0.07 * 9.4 ± 0.6 1.51 ± 0.08 * 1.07 ± 0.04 1.05 ± 0.08 1.06 ± 0.02 0.76 ± 0.05 * 0.97 ± 0.03 * V̇CO2 (L/min) RER V̇E/V̇O2 V̇E/V̇CO2 RPE 50 ± 4 46 ± 3 10 ± 0 * 42 ± 3 40 ± 2 10 ± 0 40 ± 3 41 ± 2 4 ± 1 * * * Data are means ± SE. HR, heart rate; SV, stroke volume; CO, cardiac output; MAP, mean arterial pressure; V̇E, minute ventilation; V̇O2, oxygen consumption; V̇CO2, carbon dioxide production; RER, respiratory exchange ratio; V̇E/V̇O2, ventilatory equivalent for O2; V̇E/V̇CO2, ventilatory equivalent for CO2; RPE, rating of perceived effort. * denotes significant difference from HFpEF; P < 0.05. CHAPTER 5 CONCLUSION 118 The 1st study examined the influence of central motor drive on the excitability of cortical output cells and spinal motoneurons projecting to the quadriceps during two different exercise modalities, namely, isometric knee extension and cycling exercise. The findings of this investigation demonstrate that the excitability of cortical and spinal motoneuronal projections to the knee extensors during both isometric single-leg kneeextension and cycling exercise are augmented by increases in central motor drive. This facilitation might primarily be mediated by increases in the excitability of the motoneuron pool. The 2nd study sought to elucidate age-related differences in the development of fatigue during whole body and single joint exercise. Compared to their younger counterparts, habitually active older individuals exhibit more central and peripheral fatigue during exercise against a given external load. However, during physical activities performed at the same relative intensity, younger individuals develop more central fatigue with peripheral fatigue developing similarly across ages. Thus, normalizing for relative exercise intensity offsets the age-related difference in fatigue observed during exercise performed at the same absolute intensity. Finally, the amount of active muscle mass appears to have little influence on revealing the impact of aging on exerciseinduced muscle fatigue. The 3rd study sought to quantify the impact of HFpEF on the development of neuromuscular fatigue during exercise involving a small muscle mass. Patients with HFpEF are characterized by an exacerbated development of central and peripheral fatigue during dynamic exercise involving a small muscle mass. This impairment is largely attributable to disease-related peripheral abnormalities including skeletal muscle oxygen 119 supply and utilization, and likely intrinsic muscle characteristics. As neuromuscular fatigue plays a key role in determining exercise intolerance and functional capacity, pharmaceutical treatment strategies and physical rehabilitation programs may benefit from targeting these peripheral abnormalities in HFpEF. In summary, this dissertation has explored exercise-induced alterations within the neuromuscular system and the corticospinal pathway in health and disease. The findings contribute to a better understanding of alterations within the corticospinal pathway and the development of fatigue during exercise in older individuals and patients with HFpEF.