LOW FREQUENCY FATIGUE IN ENDURANCE TRAINED, SEDENTARY, AND SPINAL CORD INJURED SUBJECTS EDWARD THOMAS MAHONEY

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1 LOW FREQUENCY FATIGUE IN ENDURANCE TRAINED, SEDENTARY, AND SPINAL CORD INJURED SUBJECTS by EDWARD THOMAS MAHONEY (Under the direction of Dr. Kevin McCully) ABSTRACT This study examined low frequency fatigue (LFF) in endurance trained (ET) and sedentary (SED) able-bodied subjects, and in individuals with spinal cord injury (SCI). ET and SED performed two separate neuromuscular electrical stimulation (NMES) protocols to evoke fatigue of the quadriceps of one thigh (experimental leg) with the un-fatigued leg as a control. Protocol 1 ( 15 Min ) lasted 15 min and the duty cycle was 33%. For protocol 2, fatigue in SED was matched to ET during the 15 min protocol ( Low Matched ) while fatigue in ET was matched to the SED 15 min protocol ( High Matched ). Force was assessed at 20 Hz (P20) and 100 Hz (P100) and the ratio of P20/P100 was used to evaluate LFF in both thighs before and up to 24 hours following fatigue. The SCI group performed only one protocol in which fatigue was matched to SED during the 15 min protocol, and evaluation of LFF was the same. Results indicated that SED had a greater magnitude of LFF compared to ET with the 15 Min (p<0.001) and High Matched (p<0.020) comparisons. The ET group did not recover faster than the SED group for any of the comparisons. Muscle pain 24 hours after the fatigue tests may have affected LFF values. For SCI, the magnitude of LFF was not significantly different compared to SED. Recovery of LFF was faster in SED compared to SCI in both the experimental (p<0.001) and control leg (p<0.001). SCI did not recover from LFF over 24 hours in either leg. When LFF values in the experimental leg were corrected for LFF values in the control leg, no difference in recovery existed between SED and SCI (p=0.064). In summary, ET had less LFF than SED even when total fatigue was matched, suggesting that ET muscle is more protected from LFF. Although ET did not recover faster from LFF, other factors such as muscle injury may make interpretations of recovery difficult. When paralyzed muscle is stimulated sufficiently, LFF will be substantial for at least 24 hours. In addition, assessing LFF with NMES in SCI causes a progressive increase in LFF, which is likely due to muscle injury. INDEX WORDS: Low frequency fatigue, electrical stimulation, spinal cord injury, endurance training, calcium impairment.

2 LOW FREQUENCY FATIGUE IN ENDURANCE TRAINED, SEDENTARY, AND SPINAL CORD INJURED SUBJECTS by EDWARD THOMAS MAHONEY B.S. Ithaca College, 1995 M.A. University of Georgia, 1998 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2006

3 2006 EDWARD THOMAS MAHONEY All Rights Reserved

4 LOW FREQUENCY FATIGUE IN ENDURANCE TRAINED, SEDENTARY, AND SPINAL CORD INJURED SUBJECTS by EDWARD THOMAS MAHONEY Major Professor: Committee: Kevin McCully Gary A. Dudley Rod Dishman Kirk J. Cureton Patrick L. Jacobs Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2006

5 DEDICATION I dedicate this work to my wife, Melanie who has always been supportive and is my pillar of strength. Melanie, I love you and I am proud to be your husband. Also, I dedicate this to Dr. Gary A. Dudley and his family as they have all faced extreme challenges and hardships over the past 4 years. When most individuals would have given up due to these overwhelming circumstances, Dr. Dudley continues to be dedicated to his work, and more importantly, his family. He has the heart of a lion and an unending drive to acquire knowledge and achieve excellence. He is a true inspiration to me, and to all that know him. iv

6 ACKNOWLEDGMENTS I would like to thank Dr. Kevin McCully for all the time you spent with me on this project and for your guidance and support throughout my doctoral degree. I have enjoyed working with you very much and hope that we might collaborate in the future. Dr. Gary Dudley, thank you for giving me the opportunity to work in your lab and to learn from you over the past 4 years. You are a true inspiration to all and your scientific knowledge is second-to-none. Dr. Kirk Cureton and Dr. Rod Dishman, thank you for your input and guidance on this project and for all the valuable information I learned in your classes. To my former mentor, Dr. Patrick Jacobs, thank you for your guidance on this project. More importantly, thank you for all the time you spent with me when I first started learning about individuals with SCI. You are the first person who sparked my interest in SCI research and I am grateful for all that I learned from you over the past years. Also, I would like to thank Dr. Debbie Backus at Shepherd Center for facilitating my data collection of SCI subjects and for all her help related to this project. I would like to thank Mrs. Dudley for her amazing strength over the past 4 years. You and your family will always be in our prayers. Also, our lab, and practically the entire building, would like to thank you for the almost unending supply of snacks to fuel our brains and add mass to our bodies! I would like to thank Timmy Stat Master Puetz for all of his help with my statistical analyses and for his friendship. Also, I would like to thank all my research subjects for participating in my study. To the lab members, Chris Elder, Tracey Tricky Kendall, and Chris Black thanks for all the good times in the lab and for your help with this project. To my brother v

7 Dave, I would like to thank you for your support and friendship over the past years and for all the laughs we have had throughout our lives. Lastly, I would like to thank my wife, Melanie for all her support and patience with me over the past 4 years. I would have never made it without you. Thank you for keeping me positive and for believing in me. I am excited to begin a new chapter of life with you in beautiful Louisville, KY. vi

8 TABLE OF CONTENTS Page ACKNOWLEDGEMENTS...v LIST OF TABLES... ix LIST OF FIGURES...x CHAPTER I. INTRODUCTION...1 Purpose...4 Specific Aims...4 Hypotheses...4 Significance of the Study...5 Limitations of the Study...6 II. REVIEW OF LITERATURE...8 Muscle Fatigue...8 Fatigue and Endurance Training...8 Calcium Related Fatigue...9 Training Status and Low Frequency Fatigue (LFF)...12 Effect of Fiber Type and Metabolic Fatigue on LFF...13 Muscle Injury and LFF...14 Surface Neuromuscular Electrical Stimulation (NMES)...16 Muscle Fatigue and Spinal Cord Injury (SCI)...17 vii

9 Low Frequency Fatigue and SCI...17 References...19 III. LOW FREQUENCY FATIGUE AFTER ELECTRICALLY EVOKED CONTRACTIONS IN TRAINED AND UNTRAINED SUBJECTS Abstract...26 Introduction...27 Methods...28 Results...35 Discussion...39 References...44 IV. LOW FREQUENCY FATIGUE IN INDIVIDUALS WITH SPINAL CORD INJURY...55 Abstract...57 Introduction...58 Methods...60 Results...66 Discussion...70 References...76 V. SUMMARY AND CONCLUSIONS...88 References...91 viii

10 LIST OF TABLES Page Table 3.1: Subjects characteristics...48 Table 3.2: Muscle pain ratings in the experimental leg in endurance trained and sedentary subjects 24 and 48 hours after completion of fatigue tests...55 Table 4.1: Individual and mean data for spinal cord injured participants...80 Table 4.2: Mean subject characteristics for SCI and able-bodied groups...81 ix

11 LIST OF FIGURES Page Figure 3.1: Percent reduction in force-time integrals during fatigue protocols in endurance trained and sedentary subjects...49 Figure 3.2: Representative force tracings at 20 Hz and 100 Hz for one sedentary participant preand 1-hour post-fatigue...50 Figure 3.3: 100 Hz force values (% initial) for 15 Min (a), High Matched (b) and Low Matched (c) conditions in ET and SED Figure 3.4: Control leg LFF values over 24 hours post-fatigue in ET and SED for the 15 Min condition...52 Figure 3.5: Magnitude of LFF for 15 Min (a), High Matched (b) and Low Matched (c) conditions in ET and SED over 1-hour post-fatigue...53 Figure 3.6: Recovery of LFF for 15 Min (a), High Matched (b) and Low Matched (c) conditions in ET and SED over 24 hours post-fatigue...54 Figure 4.1: Percent reduction in force-time integrals during fatigue protocols in spinal cord injured and able-bodied subjects...82 Figure 4.2: Representative force tracings at 20 Hz and 100 Hz for one spinal cord injured participant pre- and 1-hour post-fatigue Figure 4.3: 100 Hz force values (% initial) after a fatigue test for the experimental (a) and control (b) leg in SCI and able-bodied over 24 hours post-fatigue...84 x

12 Figure 4.4. Magnitude (a) and recovery of LFF (b) in the experimental leg of SCI and ablebodied subjects Figure 4.5. Recovery of LFF in the control leg over the 24-hour post-fatigue period in SCI and able-bodied subjects...86 Figure 4.6. Recovery of LFF in the experimental leg when statistically adjusted for LFF in the control leg of SCI and able-bodied subjects...87 Figure 5.1. The mechanisms responsible for force loss along the Injury/Fatigue continuum...93 xi

13 CHAPTER I INTRODUCTION Extensive literature exists regarding muscle fatigue (6, 21, 44, 45, 48, 59). Although muscular fatigue has been examined following different modes and intensities of exercise in many different subject populations, very small proportions of these studies have examined the mechanisms involved in the fatigue. For example, many studies have reported that fatigue is greater in individuals with spinal cord compared to able-bodied subjects, but few studies have tried to determine what factors are responsible for the enhanced fatigue in those with paralysis. Although the most widely known mechanisms of fatigue are usually thought to be metabolic in nature, the potential role that calcium may play in fatigue cannot be disregarded. The uptake and release of calcium from the sarcoplasmic reticulum (SR) are important contributors to producing and maintaining force (2, 3, 19). Many studies have demonstrated that muscular force can be reduced for several hours and may be related to disruptions in excitationcontraction coupling (19, 72-74). However, studies directly measuring calcium levels with muscular fatigue are typically invasive and are usually performed in animal models. In humans, it is possible to approximate the impairment of calcium release following muscular activity by examining low frequency fatigue (LFF), a well-documented phenomenon (17, 62, 67). Low frequency fatigue is defined as a preferential loss of force at low stimulation frequencies (ie. 20 Hz) compared to high frequencies (ie. 100 Hz). Low frequency fatigue is commonly assessed by the ratio of force produced at low and high activation frequencies following exercise (63, 66). It is characterized by a slow rate of force recovery and persistence of 1

14 reduced force in the absence of electrical or metabolic disturbances and has been shown to last 6-8 hours or longer (17, 31, 63). It is well established that aerobic athletes fatigue less during contractions than their sedentary (SED) counterparts (27, 42, 44, 68). Despite this, few studies have measured the various mechanisms related to the differences in fatigue between these two groups. However, studies have shown that endurance trained (ET) athletes recover faster from metabolic fatigue because their muscles are better equipped to buffer hydrogen ions and resynthesize phosphocreatine and ATP levels (27, 44) due to high muscle oxidative capacity. However, the question of how ET affects the long lasting recovery of muscular force (ie. LFF) due to impairment in excitation-contractions coupling has not been fully examined. Low frequency fatigue has been reported to be most dramatic in fast-twitch muscle fibers (51, 53), as well as when prior muscle injury has occurred (13, 34, 46, 53). Highly trained endurance athletes display exceptional muscular endurance and typically have greater percentages of slow-oxidative muscle fibers (56) when compared to SED. These slow-oxidative muscle fibers may be less susceptible to contraction-induced muscle injury (22, 38). Also, it appears that endurance training itself may provide some protection (18). If ET individuals, in fact have greater percentages of slow-oxidative fibers, and are more protected from contraction induced muscle injury, then it is possible that they would be less prone to LFF. Independent of muscle injury, force loss observed during a bout of muscular contractions due to metabolic factors has been shown to affect LFF (42, 50). Pronounced fatigue causes large increases in inorganic phosphate and hydrogen ions. It is hypothesized that high levels of inorganic phosphate may be taken up into the SR, where it may precipitate with calcium (23). 2

15 The formation of calcium phosphate would lower free calcium concentrations in the SR, thus reducing calcium release. A study by McCully et al. (42) determined that protocols that elicit high levels of fatigue, and therefore metabolic byproducts, can increase the magnitude of LFF observed during recovery. With regard to ET and SED individuals, previous literature would indicate that when these two groups perform identical protocols that SED would incur greater metabolic fatigue and force loss. For these reasons, it seems plausible that SED individuals would have greater LFF following contractions, compared to ET subjects. Surface neuromuscular electrical stimulation (NMES) is commonly used in testing of able-bodied subjects as well as in those with neuromuscular disorders to facilitate contraction of weak or paralyzed skeletal muscle (5, 11, 39). Activating skeletal muscle for the purpose of functional movements and exercise through NMES is challenging as skeletal muscle fatigue can occur rapidly. The profound fatigue that occurs with this modality, is likely due to altered motor unit recruitment patterns (20, 35, 36), as well as synchronous activation of a given motor neuron pool (1, 52). It is well established that the use of NMES requires greater energy demand and consequently causes more fatigue when compared to similar voluntary efforts (29, 35, 69). Muscle fatigue has been reported to be greater in individuals with spinal cord injury (SCI) when compared to able-bodied subjects (5, 10, 30, 48). The most widely supported mechanism explaining the high levels of fatigue observed in SCI subjects has been attributed to the increased proportion of fast-twitch fibers (8, 25). To our knowledge, few studies have thoroughly examined LFF in individuals with SCI. Since there is a somewhat complete transformation from slow to fast contractile machinery with chronic SCI, as well as an increased 3

16 risk of contraction-induced muscle injury (5), it seems plausible that paralyzed muscle would be more susceptible to LFF than normally loaded muscle. Purpose The general aim of this study is to better understand the mechanisms of muscle fatigue, in particular in individuals who are extremely fit, as well as in those who are severely deconditioned. This will be accomplished by measuring the magnitude and recovery of low frequency fatigue resulting from two NMES-evoked fatigue protocols in endurance trained (ET) and sedentary (SED) able-bodied subjects, as well as in individuals with spinal cord injury (SCI). Low frequency fatigue (LFF) will be measured for up to 24 hours in each of these groups after performing two protocols which induce different amounts of fatigue. This study will determine if the total amount of force loss during a fatigue protocol affects the LFF response or whether additional factors play a role. Also, this study will determine if use of a control leg (un-fatigued leg) when examining LFF is warranted. Specific Aims 1. In ET and SED subjects, the magnitude and recovery rates of LFF were evaluated in response to separate NMES-evoked protocols designed to initiate two levels of fatigue. 2. In SCI and SED subjects, the magnitude and recovery rates of LFF were evaluated in response to a separate NMES-evoked protocol designed to match force loss in both groups. Hypotheses 1a. ET will have less magnitude and faster recovery of LFF compared to SED following the performance of an identical fatigue protocol (15 Min). 4

17 1b. ET will have less magnitude and a faster recovery of LFF compared to SED following the performance of fatigue protocols designed to match relative force loss (High and Low Matched conditions). 2. SED able-bodied controls will have less magnitude and faster recovery of LFF compared to SCI following the performance of fatigue protocols designed to match relative force loss between groups. Significance of the study It is unknown if calcium related fatigue (ie. LFF) can be reduced with endurance training, even though it is well known that overall muscle fatigue is greatly reduced. SCI is a condition associated with enhanced muscle fatigue and increased susceptibility to contraction-induced muscle injury, both of which have been shown to enhance LFF. The increased muscle fatigue/injury in SCI limits the ability to use NMES as a therapeutic tool. This study is the first to thoroughly examine LFF in subjects with SCI, as well as how training status influences the LFF response. An important aspect of this study will be to monitor the magnitude and recovery of LFF after a fatiguing bout of exercise. This study is unique in that it tested if enhanced fatigue with deconditioning (SED and SCI) is related to a large magnitude and prolonged recovery of LFF, and whether electrical stimulation protocols causing different levels of fatigue will affect this response. Using various fatigue tests allowed examination of LFF after the same number of stimulations and after the same percentage of force loss between groups. By performing this study, more was learned about the importance of LFF in humans after use of NMES. Low frequency fatigue has clinical implications in that individuals with SCI, or other neuromuscular disorders, may use low-frequency NMES several times a day for 5

18 rehabilitation to make weak or paralyzed muscle contract. The results of this study may aid clinicians in designing appropriate electrical stimulation protocols for rehabilitation. Limitations of the study This study assessed LFF, a phenomenon thought to be due to impaired calcium release during muscular contractions. This study, however, did not directly measure calcium release rates and was not designed to determine the role that various mechanisms may have on LFF (ie. muscle injury that may occur). The ratio of force produced at low and high frequency (P20/P100) was assessed after a fatigue protocol to quantify LFF. The magnitude and recovery of LFF may give a rough idea of how much excitation-contraction coupling is impaired, but LFF data was not used to generalize calcium uptake or release rates. Another potential limitation of this study is that subjects had to re-enter the force chair several times throughout the 24-hour post-fatigue period, which may lead to increased variability in the force measurements. To limit variability of testing measures, a control leg was utilized in the research design. In addition, subjects were asked to remain relatively inactive over the course of the day and the electrodes were traced to ensure the same placement for the 24-hour assessment of LFF, or at any other time the electrodes were taken off during testing. It has been reported that LFF is more pronounced when prior muscle injury has occurred. This is not a large concern for the able-bodied subjects as research has shown that these types of contractions do not typically induce muscle injury. However, NMES-evoked isometric contractions are capable of eliciting muscle injury in those with long-term SCI. Although we tried to limit muscle injury by keeping force values low, we did not quantitatively assess muscle injury, which could potentially limit the interpretation of the results of this study, especially in 6

19 the SCI group. However, if NMES-evoked force returns to baseline values 24 h post-fatigue, then we can assume little, or no injury has occurred. 7

20 CHAPTER II REVIEW OF LITERATURE Muscle fatigue Skeletal muscle fatigue is defined as the failure to maintain the required or expected power output (16). Skeletal muscle fatigue ordinarily occurs due to a number of plausible theories including failure at the neuromuscular junction, decreased calcium release and reuptake, buildup of metabolic byproducts, and central fatigue (21). In addition, factors outside the motor unit, including inadequate blood flow, may contribute to muscle fatigue. Force recovery following fatigue is made up of three distinct components (45). With the first component, muscular force recovers quickly (within seconds) and is likely related to disruption in electrical excitability of the muscle membrane or buildup of K + in the transverse tubules, possibly related to the disruption of Na + -K + adenosinetriphosphatase pump function. The second component is due to the build-up of various metabolites produced during exercise, which recover somewhere between 3-20 minutes. The third component of fatigue, which has been demonstrated to last up to 6-8 h and potentially longer, is likely related to disruption of excitation-contraction coupling (calcium impairment), and force loss is most severe when muscle actions are elicited at low frequencies (ie.1-20 Hz). Fatigue and endurance training It is well established that endurance trained (ET) athletes fatigue less and recover quicker than their sedentary (SED) counterparts (27, 42, 44, 68). Theriault et al. (68) examined resistance to fatigue in the knee extensor muscles of active and ET individuals. Subjects performed 25 8

21 maximal voluntary isometric contractions, each lasting 10 s with 5 sec rest between contractions and demonstrated a significant difference in the amount of fatigue between both groups. This study demonstrates that ET fatigue less with intermittent-type exercise, which is likely due to faster recovery between contractions. McCully et al. (44) examined muscle metabolism in controls and long-distance runners using 31 P magnetic resonance spectroscopy. Phosphocreatine was measured during and following 5 min of repeated plantar flexion of the calf muscles. The maximal rates of PCr resynthesis were nearly twice as fast for long-distance runners as compared to controls (64.8 vs mmol/min/kg muscle). The differences shown here are consistent with literature demonstrating that long-distance runners have faster recovery of metabolites than controls, which is directly due to higher oxidative capacity. Extensive literature exists regarding muscle fatigue related to the buildup of metabolic byproducts, namely inorganic phosphate and hydrogen ions, and their mechanisms of action are quite well known (44, 45, 69-71). Calcium related fatigue (LFF) The proposed project will not examine metabolic fatigue but will be more focused on examining the long-lasting fatigue related to calcium impairment. Although some researchers believe that loss of muscular force is almost completely due to the accumulation of metabolic byproducts, it is becoming clear that calcium impairment may play a larger role in fatigue than previously thought. The uptake and release of calcium from the sarcoplasmic reticulum (SR) are important contributors to producing and maintaining force. It has been demonstrated that the onset of muscle fatigue may be related to the inability of the SR to adequately release and sequester calcium (19). Also, accumulation of metabolic byproducts themselves might cause reductions in calcium release from the SR (2). Insufficient delivery of calcium to the myofilaments would lead to reduced force output and may be caused by impaired excitation- 9

22 contraction coupling, changes in SR calcium concentrations, or by transient modifications of the SR calcium channel (19). The strongest evidence points to transient modification of the SR calcium channel with fatigue, which reduces its opening probability and decreases calcium release into the myoplasm. Data from animal models indicate that when intracellular calcium [Ca +2 ] i concentration are elevated, general calcium homeostasis becomes impaired and calcium release is subsequently reduced (14, 37, 74). Also, it has been reported that high levels of [Ca +2 ] i are capable of activating proteases that lead to cellular damage. Therefore, reducing calcium release with fatigue would favor a lower calcium environment (due to continued sequestration of calcium), which may act to maintain cellular integrity by limiting calcium-mediated damage (9). Pronounced fatigue causes large increases in inorganic phosphate and hydrogen ions. It is hypothesized that high levels of inorganic phosphate may be taken up into the SR, where it may precipitate with calcium (23). The formation of calcium phosphate would lower free calcium concentrations in the SR, thus reducing calcium release. However, the ensuing impairment in calcium release would preferentially affect muscular forces at low activation frequencies. Higher frequencies, however, are capable of overcoming such impairments and can produce saturating levels within the myoplasm, thus blunting reductions in muscle force. From such studies, it is becoming increasingly clear that impaired calcium handling is an important mechanism involved in muscular fatigue. Studies directly measuring calcium (concentrations, release/reuptake rates, receptor and channel activity, etc.) with muscular fatigue are typically invasive and are usually performed in animal models. Measuring calcium handling in humans is not as simple. In humans, we can obtain muscle biopsies to measure calcium release, uptake, and Ca +2 -adenosinetriphosphatase activity with various protocols. From force tracings, we can obtain crude estimates of calcium 10

23 kinetics by examining rise and relaxation times during contractions. In addition, it is possible to approximate the magnitude of calcium release impairment following muscular activity by examining low frequency fatigue (LFF), a well-documented phenomenon. Low frequency fatigue is defined as a preferential loss of force at low stimulation frequencies (ie. 20 Hz) compared to high frequencies (ie. 100 Hz). Low frequency fatigue is commonly assessed by the ratio of force produced at low and high activation frequencies following muscular fatigue (63, 66, 67). It is characterized by a slow rate of force recovery and persistence of reduced force in the absence of electrical or metabolic disturbances and has been shown to last 6-8 hours or longer (17, 31, 63). After a bout of fatiguing exercise, force produced at low and high electrical stimulation frequencies should both be affected by metabolic fatigue (41) which is known to subside ~20 min following exercise (45). High frequency force is less affected by this calcium related fatigue, whereas low frequency force appears to be preferentially suppressed. Low frequency fatigue is likely due to impaired excitation-contraction coupling with evidence specifically pointing to blunted calcium release from the SR at low stimulation frequencies (17, 45, 73). Chin et al. (15) have reported that the reduction in calcium release with LFF is primarily due to an elevation in the [Ca +2 ] i -time integral, which represents the cumulative increase in [Ca +2 ] i above resting levels for series of tetanic contractions. A common misconception is that LFF is only caused by low frequency evoked muscle contractions. Studies have elicited LFF with voluntary contractions, (41, 52, 63) as well as with both high ( Hz) and low frequency (1-30 Hz) electrically induced contractions (32, 52, 74). However, Ratkevicius et al. (52) have demonstrated that electrically evoked contractions actually cause more LFF than do voluntary contractions and longer contractions (30 sec) cause 11

24 more pronounced LFF, as compared to shorter ones (5 sec). Although high frequency fatigue protocols will elicit LFF, there is some evidence that lower frequencies cause a more pronounced LFF (7). In addition to longer individual muscle contractions, longer duration fatigue protocols themselves have been shown to cause greater LFF (4). In summary, LFF is most pronounced following longer individual contractions which are electrically evoked, and that are performed within fatigue protocols that are of longer duration. Training status and LFF A question that has not been fully examined is how training status may influence LFF after fatiguing bouts of exercise. Since LFF has been attributed to impaired calcium release from the SR, it has been suggested that high intensity training may help to partially compensate for this impairment. A study by Willems & Stauber (75) examined LFF in rat plantar flexors with 6 weeks of high-speed eccentric resistance training. They showed significantly smaller reductions in the P20/P100 ratio in trained vs. control muscles after an electrically stimulated fatigue test. Although no calcium related measures were performed in this study, they attributed the reduction in LFF in the resistance-trained muscles to increase release of calcium from the SR. In support of this, Ortenblad et al. (49) demonstrated that 5 weeks of sprint training in men led to an enhanced rate of peak SR calcium release, whereas calcium sequestration function was not changed. Although calcium release may be enhanced with high intensity trained (sprint and resistance training), it is likely that muscle fiber types do not get faster. If anything, this type of training may cause a fiber type shift from type IIb to IIa, which is known to occur when sedentary individuals become active (68). Something inherent about this fiber type shift may cause reductions in LFF as it has been reported that fast-fatigable fibers are more prone to LFF than fast-fatigue resistant fibers (51). Although high intensity training appears to reduce LFF 12

25 following a bout of exercise, there is not much information on how endurance training affects LFF. Effects of fiber type and metabolic fatigue on LFF Following muscular contractions, LFF is evident in both fast and slow twitch muscle fibers of animals, as well as humans (33). However, it has been reported that fast-glycolytic fibers demonstrate greater LFF following types of muscular contractions than do fast-oxidative fibers (51, 53). Powers & Binder (51) reported that fatigue resistant motor units from cat flexor digitorum muscles exhibited less pronounced LFF than fast-intermediate and fast-fatigable motor units after a series of electrically stimulated contractions. Also, they demonstrated a significant positive correlation (r = 0.611) between tetanic tension of individual motor units and the magnitude of LFF, indicating the greatest LFF was observed in the largest motor units. Force loss observed during a bout of muscular contractions due to metabolic factors has been shown to affect LFF (42, 50). In the cat gastrocnemius muscle, Parikh and colleagues (50) examined LFF after protocols of 2, 5, 10, 20 and 50 concentric contractions at optimal muscle length. As expected, they demonstrated that force loss after 50 concentric contractions was greater than the trials where fewer contractions were performed. They demonstrated that as the number of the contractions in a protocol increased, force loss was greater, and LFF was more pronounced after exercise. McCully et al. (42) used an in vivo rat muscle model to examine the potential role that metabolic byproducts might have on calcium related fatigue (ie. LFF). They examined muscular force and metabolic byproducts during and following bouts of electrical stimulation. Two stimulation protocols were used: 1) high intensity stimulation followed by medium intensity stimulation (High Group), and 2) low intensity stimulation followed by medium intensity 13

26 stimulation (Low Group). Metabolic fatigue was based on concentrations of inorganic phosphate and LFF was assessed as the relative decline of force at low compared to high stimulation frequencies. During the initial stimulation period, the High Group had greater metabolic fatigue and LFF compared to the Low Group. During the second stimulation and recovery period, the High group demonstrated no difference in metabolic fatigue but had significantly greater LFF. They determined that protocols that elicit high levels of fatigue, and therefore metabolic byproducts, can increase the magnitude of LFF observed during recovery. With regard to ET and SED individuals, previous literature would indicate that when these two groups perform identical protocols that SED would incur greater metabolic fatigue and force loss. It is possible that greater metabolic fatigue observed in SED subjects may lead to a greater magnitude of LFF following contractions, compared to ET subjects. Muscle injury and LFF Low frequency fatigue has been reported to be greater when prior muscle injury has occurred (13, 34, 46, 53). Since the origin of LFF has been attributed to disruption in the excitation-contraction coupling process, studies have demonstrated that muscle injury can impair calcium release/reuptake rates and affect muscular force, most predominately at low activation frequencies (9, 46). Rijkelijkhuizen et al. (53) examined the effects of 40 isometric, concentric, or eccentric muscle actions on LFF in different fibers types of the rat medial gastrocnemius. Seventy minutes post-exercise, LFF was more severe with eccentric compared with isometric and concentric contractions. Of the three contraction types, eccentric actions cause the greatest disruption to muscle fibers (43, 47). This study indicates that more injurious bouts of exercise demonstrate greater LFF. Newham et al. (46) used a bench step test to initiate muscle injury using concentric contractions in the quadriceps of one leg (step up leg) and eccentric contractions 14

27 in the other (step down leg). Regardless of contraction type, this protocol caused pronounced LFF, indicated by a reduction in the P10/P50 ratio, which was still significantly suppressed 24 hours following bench stepping. However, the leg that performed the eccentric contractions had a significantly larger reduction in P10/P50 as compared to the concentric leg, for up to 5 hours post-exercise. These studies indicate the dramatic effect that muscle injury can have on muscle force at low activation frequencies. Highly trained endurance athletes display exceptional muscular endurance and typically have greater percentages of slow-oxidative muscle fibers (56) when compared to SED. In addition to less pronounced LFF, these slow-oxidative muscle fibers have been reported to be less susceptible to contraction-induced muscle injury (22, 38). Also, it has been reported that endurance training, specifically running, seems to provide protection from muscle injury due to the eccentric component involved with this exercise modality (18, 57). If ET individuals, in fact have greater percentages of slow-oxidative fibers, and are more protected from contraction induced muscle injury, then it is possible that they would be less prone to LFF following a bout of exercise designed to injure skeletal muscle. However, a study by Skurvydas et al. (62) showed no differences in LFF between long-distance runners, sprinters and untrained men after stretchshortening exercise, which consisted of 100 maximal drop jumps. They may have failed to show differences between groups due to the fact that LFF was measured in the muscles of the thigh, but other muscle groups were likely involved during exercise. Also, drop jumps performed may not have induced substantial fatigue and/or muscle injury solely to the quadriceps muscle, potentially not evoking enough LFF to distinguish differences between groups. 15

28 Surface neuromuscular electrical stimulation (NMES) The use of surface neuromuscular electrical stimulation (NMES) is commonly used in testing of able-bodied subjects as well as in those with neuromuscular disorders to facilitate contraction of weak or paralyzed skeletal muscle. Activating skeletal muscle for the purpose of functional movements and exercise through NMES is challenging as skeletal muscle fatigue can occur rapidly. The profound fatigue that occurs with this modality, is likely due to altered motor unit recruitment patterns (20, 35, 36), as well as synchronous activation of a given motor neuron pool (1, 52). It is well established that the use of NMES requires greater energy demand and consequently causes more fatigue when compared to similar voluntary efforts (28, 35, 69). Utilizing high frequencies ( Hz) of NMES leads to rapid onset of fatigue but force typically returns to baseline values fairly quick during recovery. Alternatively, low frequency NMES typically leads to less fatigue during contractions but recovery of force is prolonged (17, 31, 63). It is believed that fast-twitch fibers are activated to a greater extent with NMES than during voluntary activation at matched submaximal workloads (35, 69). The enhanced force loss with NMES is thought to be due to the greater myofibrillar adensosine triphosphatase rates in these fast-twitch muscle fibers as well as their rapid loss of phosphocreatine stores (65). Research has focused on trying to limit skeletal muscle fatigue with the use of NMES systems in able-bodied subjects as well as those with neuromuscular diseases. One such approach has been experimentation with variable frequency trains (55, 64), which alter the pulse frequency within a train of stimulation. Variable-frequency trains may show promise in future NMES systems, although recent data shows that the efficacy of this stimulation in spinal cord injured (SCI) patients appears to be limited (58). In summary, exercise evoked via NMES leads to greater 16

29 levels of fatigue, which contributes to low levels of work typically reported with traditional NMES-evoked training, and generally translates into limited improvements in fitness and health. Muscle fatigue and SCI Muscle fatigue has been reported to be greater in individuals with SCI, as compared to able-bodied subjects (5, 12, 30). The most widely supported mechanism explaining the high levels of fatigue observed in SCI subjects has been attributed to the increased proportion of fasttwitch fibers, which have greater ATP turnover rates compared to slow twitch fibers. Slow to fast fiber conversion has been shown to occur after 1-2 years post-injury with increased expression of myosin heavy chain IIa and IIx (8, 25), as well as faster contraction speeds (24, 54). Muscle biopsy data from m. tibialis anterior from SCI subjects 2-11 years post-injury indicate significantly less slow-twitch fibers than controls (69% vs. 14%, respectively) and smaller mean fiber cross-sectional area (40). It is reported that a near complete conversion from slow to fast muscle occurs over a period of approximately 70 months post-injury (8). Along with fiber type changes, studies have demonstrated decreases in mitochondrial size as well as reduced oxidative enzyme activities (26, 40). Over time, the paralyzed skeletal muscle contains smaller fibers that have a greater energy demand (greater contractile speed) and a lower capacity to supply it, leading to greater muscle fatigue. These factors likely account for some of the difference in fatigability between SCI and able-bodied, with other factors possibly including altered calcium handling and/or muscle injury occurring during the onset of muscular contractions. Low frequency fatigue and SCI Although many studies have examined fatigue during contractions in SCI, few have examined force for several hours after a bout of NMES-evoked exercise. Since there is a 17

30 somewhat complete transformation from slow to fast contractile machinery with chronic SCI, as well as an increased risk of contraction-induced muscle injury (5), it seems plausible that paralyzed muscle would be more susceptible to LFF than able-bodied muscle. To our knowledge, few studies have examined LFF in individuals with SCI. Shields et al. (61) examined low frequency force loss and recovery in the soleus muscle of chronic (> 3 years post-injury) and acute SCI (< 5 weeks post-injury). The fatiguing NMES protocol consisted of 330 ms trains delivered every second for 3 minutes. During the fatigue protocol they measured twitch (1Hz) and tetanus (20Hz) force every 30 seconds during fatiguing contractions and at 5 min postexercise. Immediately following exercise, peak twitch and tetanus torque was reduced ~80% and 75%, respectively in the chronic SCI compared to only ~ 14% and 16% in acute SCI group. Twitch and tetanus force in acute SCI had fully recovered within 5 minutes whereas chronic SCI force had only recovered to ~65% and 60% of pre-fatigue values at 1 and 20 Hz, respectively. This study, however did not actually measure LFF because high frequency force was not assessed. Also, the fatigue protocol did not evoke LFF in the acute SCI group because low frequency force (1 and 20 Hz) had recovered fully within 5 minutes. Another study by Shields group (60), which was nearly identical to the aforementioned study, was performed on 8 individuals with SCI (7 chronic, 1 acute) but assessed force at several frequencies before, immediately after, and at 5 and 15 min following a 4 min fatigue protocol. The frequencies used to assess force before and after the fatigue protocol were 1, 5, 10, 15, 20, 30, and 40 Hz. Extracting data from their graphs and calculating the ratio of force produced at 10 and 40 Hz as an indicator of LFF, yields ratios of 0.49, 0.34, 0.24, and 0.22 for before, immediately after, and 5 and 15 min following fatigue, respectively. Although this study 18

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