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Does Heat Treatment Facilitate Muscle Regrowth following Hind Limb Immobilization?

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PAGE 1

DOES HEAT TREATMENT FACILITA TE MUSCLE REGROWTH FOLLOWING HIND LIMB IMMOBILIZATION? By JOSHUA SELSBY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

PAGE 2

Copyright 2005 by Joshua Selsby

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This document is dedicated to my favorite Anatomy student.

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ACKNOWLEDGMENTS First and foremost, I would like to thank my wife for her patience and understanding. I also thank my parents who have provided much needed encouragement and support over the years. The Muscle Physiology Lab group, especially Andrew Judge, Sara Rother, Om Prakash, and Shige Tsuda, certainly receive my thanks and gratitude. I thank my committee, Scott Powers, David Criswell, and Glenn Walter, for their time and thought into this project. Lastly, I would like to thank my advisor, Stephen Dodd, who has helped guide me to the completion of this project. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES ............................................................................................................vii LIST OF FIGURES .........................................................................................................viii ABSTRACT .........................................................................................................................x CHAPTER 1 INTRODUCTION........................................................................................................1 Background...................................................................................................................1 Questions......................................................................................................................4 Hypotheses....................................................................................................................4 2 REVIEW OF LITERATURE.......................................................................................5 Significance..................................................................................................................5 Free Radicals and Oxidant Stress.................................................................................6 Free Radicals.........................................................................................................6 Sources of Free Radicals.......................................................................................7 Sources of Free Radicals During Immobilization.................................................8 Regulation of Free Radicals..................................................................................9 Skeletal Muscle and Unloading..................................................................................10 Models.................................................................................................................10 Changes with Disuse Atrophy.............................................................................12 Oxidative Stress, Antioxidants and Unloading...................................................14 Skeletal Muscle and Reloading..................................................................................15 Adaptations to Reloading....................................................................................15 Pathways of Regrowth.........................................................................................16 Reloading: Injury and Damage............................................................................17 Reloading and the Immune Response.................................................................18 Oxidant Damage..................................................................................................19 Heat Shock Proteins....................................................................................................21 Overview.............................................................................................................21 v

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Heat Shock Proteins 27, 32, and 72.....................................................................22 Heat Shock Proteins, Oxidant Stress, and Potential as a Reloading Intervention......................................................................................................24 3 METHODS.................................................................................................................28 Design.........................................................................................................................28 General Procedures.....................................................................................................29 Immobilization and Reloading............................................................................29 Heat Treatment....................................................................................................30 Muscle Removal and Sample Preparation...........................................................31 Biochemical Procedures.............................................................................................32 Western Blot........................................................................................................32 Glutathione Peroxidase........................................................................................33 Glutathione Reductase.........................................................................................34 Catalase................................................................................................................34 Superoxide Dismutase.........................................................................................35 Ferrous Oxidation Xylenol Orange...................................................................35 ELISAS................................................................................................................36 Statistical Analyses.....................................................................................................36 4 RESULTS...................................................................................................................37 Whole Body Measures................................................................................................37 Whole Muscle Measures.............................................................................................38 Oxidative Damage......................................................................................................40 Anti-Oxidant Enzymes...............................................................................................42 IGF-1 Pathway Activation..........................................................................................43 Heat Shock Proteins....................................................................................................47 5 DISCUSSION.............................................................................................................50 Muscle Mass...............................................................................................................50 Oxidative Stress and Antioxidant Enzymes...............................................................52 Heat Shock Proteins....................................................................................................55 IGF-1 Pathway............................................................................................................58 Integration...................................................................................................................61 LIST OF REFERENCES...................................................................................................64 BIOGRAPHICAL SKETCH.............................................................................................84 vi

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LIST OF TABLES Table page 4-1 Body weight changes at various time periods during this investigation. Values are shown in grams and are presented as means SEM..........................................38 4-2 Relative muscle mass, water content, and wet weight to dry weight ratio in the Con, Im, R7C and R7H groups................................................................................40 4-3 Activities of various antioxidant enzymes in the Con, Im, R7C and R7H groups...43 vii

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LIST OF FIGURES Figure page 2-1 Schematic of antioxidant scavenging system...........................................................10 3-1 Schematic of study design........................................................................................29 3-2 Schematic of protocol for one hypothetical week....................................................29 4-1 Wet muscle mass in control animals (Con), following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H)........................................................................................................39 4-2 4-hydroxy-non-enol as determined by immuno-blotting following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H)........................................................................................................41 4-3 Nitrotyrosine as determined by dot blot immuno-blotting (top) and conventional western blotting (bottom) following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H)...41 4-4 Akt content (top) and phosphor-Akt content (bottom) as determined by immuno-blotting following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H)............................................44 4-5 Total Gsk (top) and phosphorylated Gsk (bottom) content as determined by immuno-blotting following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H)..............................45 4-6 Total p70s6k (top) and phosphorylated p70s6k (bottom) content as determined by immuno-blotting following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H)..............................47 viii

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4-7 Relative hsp 27 content following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H)...48 4-8 Relative hsp 72 content following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H)...49 4-9 Hsp 32 (HO-1) content following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H)...49 ix

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DOES HEAT TREATMENT FACILITATE MUSCLE REGROWTH FOLLOWING HIND LIMB IMMOBILIZATION? By Joshua Taylor Selsby December 2005 Chair: Stephen Dodd Major Department: Applied Physiology and Kinesiology In order for skeletal muscle to return to its original state following immobilization, reloading of the muscle is necessary. Reloading is characterized by a period of increased oxidant damage to skeletal muscle macromolecules. Previous work has shown that antioxidant supplementation during reloading will decrease oxidant stress and augment the rate of muscle hypertrophy. We have previously shown that heating can provide a potent antioxidant effect. It was hypothesized that heating in conjunction with reloading would result in a faster rate of hypertrophy than reloading alone. In order to test this hypothesis, male Sprague-Dawley rats were divided into control (Con), immobilized (Im), reloaded (R7C), and reloaded heated (R7H) groups (n=10/group). Animal hind limbs in the Im, R7C and R7H group were plantarflexed and immobilized bilaterally for one week. At the conclusion of one week, Im animals were killed and reloaded animals were returned to their cages for one week. Beginning 24 hrs. x

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prior to reloading and continuing on alternating days, animals in the R7H group were heated such that the core temperature was sustained at 41.5C for 30 min. Immobilization resulted in a reduction in muscle mass that was not corrected by reloading alone; however it was significantly increased in the R7H group. In addition, oxidant stress to lipids and proteins was increased in the Im and R7C groups; however, reloading in combination with heat returned both markers to Con levels. Assessment of the antioxidant enzymes revealed that they are not responsible for the observed antioxidant effect of heating. Heat shock proteins 25, 32, and 72 were increased in the R7H group when compared to the R7C group indicating that they may be responsible for the observed antioxidant effect. Lastly, the R7C group may suffer from an IGF-1 pathway dysfunction that is corrected in the R7H group. The summation of this data is that heating augments muscle regrowth following immobilization. Further, it reduced oxidant damage via an antioxidant enzyme independent mechanism, and likely through a heat shock protein dependant mechanism. Lastly, heating corrected the IGF-1 dysfunction seen during reloading. xi

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CHAPTER 1 INTRODUCTION Background As life expectancy continues to expand, the incidence of infirmity and the frequency of immobilization will increase. In addition, our increasing emphasis on prolonged space travel will require an extended exposure to microgravity. Regardless of method, severe atrophy of the locomotor muscles will follow. Much research has been dedicated to the study of unloading and development of countermeasures to this type of muscle loss. What has been neglected, however, is the need to reload these muscles following unloading. The present study is designed to investigate a potential countermeasure to damage that occurs during the reloading process. Disuse atrophy induced via casting has been repeatedly demonstrated to reduce skeletal muscle mass, cross sectional area, and force (8-10, 22, 69, 100, 108, 112, 197, 219). Furthermore, upon reloading, the atrophied muscle undergoes damage much like that seen with a pliometric (eccentric) contraction (69, 94, 100, 227). Accompanying this damage is an increase in the oxidative stress encountered by the reloaded muscle (108, 236). It has also been shown that supplementation with an antioxidant can reduce the oxidative stress and lessen damage observed with reloading and hasten muscle regrowth (108). The concept of reloading injury as being caused by a pliometric contraction is not a new idea. In fact, in normal cage sedentary activity the ambulation cycle contains a lengthening contraction component (131). When the hind limb is immobilized in the 1

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2 shortened position, severe atrophy of the soleus occurs, both in terms of mass and length (27, 49, 186). As the soleus is the primary antigravity muscle and ambulatory muscle of the hind limb, it is not surprising that it undergoes contraction-induced injury upon reloading especially when it is considered that a shortened muscle will be required to work through the range of motion of its previous length. Furthermore, it is possible that reloading causes the muscles load-bearing capacity to be exceeded (100). Histologically this damage can be observed as irregular widening of sarcomeres, A band disruptions, and Z-line streaming (65, 184, 185, 187). Further, injured muscle may also have centralized nuclei and membrane disruptions (42, 164, 165, 223). The cause of this damage initially was thought to be mechanical in nature. Clearly a mechanical component exists as myosin and titin disruptions have been observed (221, 237). It is now apparent that a secondary oxidative stress also occurs during reloading and may exacerbate the damaged condition within the reloaded fiber (108). It seems likely that this secondary oxidative stress is occurring due to several factors involving a loss of Ca 2+ homeostasis and containment within the cell during reloading (21, 94). The elevated intracellular Ca 2+ can in turn activate Ca 2+ sensitive proteins, likely calpains, that convert xanthine dehydrogenase to the free radical producing xanthine oxidase resulting in the production of super oxide (113, 146). A second source of free radical production may be of mitochondrial origin. As the Ca 2+ levels rise, more is transported into the mitochondria via the uniporter, which has the effect of reducing membrane potential and electron transport (82, 85). It also causes an increase in electron leakage and increases superoxide production (39, 57, 85, 86). Finally, increased Ca 2+ concentrations will increase NO synthesis and the potential for ONOO production (136). The production of

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3 free radicals is made worse by the fact that the fiber already is in a state of compromised oxidant resistance (113, 122). From previous work, it can be concluded that countering this elevated oxidative stress is beneficial to muscle regrowth as antioxidant supplementation resulted in larger muscles following reloading (108). Hence, a potential mechanism to enhance muscle regrowth following disuse atrophy may be the application of a heat stress causing the subsequent over expression of heat shock proteins (hsps) and a concomitant reduction in oxidant stress. There are several features of hsps, hsp 27, 32, and 72 in particular, that make them attractive as potential candidates for this role. Together, these proteins can scavenge free radicals, assist in the folding of nascent polypeptides, refold damaged proteins, and assist in the clearance of proteins damaged beyond repair (72, 106, 120, 133, 163, 200, 211). By removing free radicals from the fiber, they are not available to cause damage. By folding nascent polypeptides the elongation phase of protein synthesis is actually enhanced resulting in a more rapid rate of protein synthesis. Refolding damaged proteins will spare them from degradation. Finally, clearing damaged proteins will prevent them from accumulating within the cell and cause a potentially cytotoxic accumulation. The notion of using a heat intervention in order to confer protection during in vivo perturbations is not new as it has repeatedly been shown to be cardioprotective (77, 98) as well as protective to skeletal muscle during an ischemia/reperfusion injury (124). Heating has also been shown to attenuate muscle atrophy both by hind limb unweighting (161) and by hind limb immobilization (197). In each of these cases, protection was attributed to the over-expression of hsps. More recently, McArdle et al. (144) were able

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4 to show that following pliometric injury, transgenic mice over-expressing hsp 72 recovered faster and did not suffer the well-characterized secondary force reduction. Additionally, transgenic mice had a higher degree of undamaged muscle when compared to wild-type. Thus, the present investigation is designed to examine whether heat stress can decrease oxidative damage found within the cell while simultaneously increasing hypertrophy during the reloading period. Questions The following questions were investigated in this study. 1. Will heating reduce oxidant damage to both lipids and proteins during reloading after immobilization? 2. Will heating cause changes in the activities of native antioxidant enzymes during reloading after immobilization? 3. Will heating accelerate the rate of muscle regrowth during reloading after immobilization? 4. Will heating cause a greater activation of signals of protein synthesis during reloading after immobilization? Hypotheses The following hypotheses were made in this study. 1. Heat treatment will reduce oxidative damage to both lipids and proteins during reloading. 2. Heat treatment will result in a reduction in antioxidant enzyme activity during reloading. 3. Heat treatment will cause an increased muscle regrowth rate following eight days of reloading. 4. Heating will cause an increased activation of protein synthesis components during reloading.

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CHAPTER 2 REVIEW OF LITERATURE Significance Muscle atrophy is a critical health concern in several disease states and has an impact on the quality of life and independent living in the elderly (156, 218). Much of this atrophy occurs due to interventions, including bed confinement and cast immobilization implemented by physicians in care of their patients. Regardless of the cause of atrophy, reloading of the muscle will be necessary in order to return to a pre-atrophy activity level. Much research has been conducted investigating mechanisms that cause atrophy. Further, a host of countermeasures have been found that appear to be effective in reducing atrophy. Comparatively little research has been conducted investigating the potential for interventions that enhance the regrowing process during subsequent muscle reloading. This review will focus on muscle reloading, building toward a hypothesis for a novel intervention during the reloading process. In order to fully appreciate changes in skeletal muscle occurring during reloading it is important to discuss several related topics as well. Oxidant stress increases during disuse atrophy and reloading; hence an understanding of oxidative stress is relevant. Secondly, it is also important to understand the condition of the muscle following disuse atrophy, as significant remodeling has occurred. Finally, as relatively little research has been conducted encompassing some topics of reloading, lengthening contraction data will also be included and discussed. This is relevant because during normal ambulation there is a lengthening contraction 5

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6 experienced by the hind limb muscles (134). Under normal conditions, the muscle has adapted to this repeated pliometric contraction and, as such, it is not damaging. Upon reloading following disuse, these repeated pliometric contractions can be quite devastating to the muscle. As the damage inflicted comes as a result of lengthening contractions, it becomes important to cull this literature of material relevant to the discussion at hand (134). Finally, this review will propose the use of heat stress and resultant over-expression of hsps as a means to augment the regrowing process. Free Radicals and Oxidant Stress Free Radicals A free radical is a molecule or atom, existing independently, with an unpaired valence electron (33, 54, 186). More specifically, reactive oxygen species (ROS) are oxygen containing free radicals, superoxide (O 2 -) and the hydroxyl radical (OH). Reactive nitrogen species (RNS) are nitrogen containing free radicals and include nitric oxide (NO) and peroxynitrate (ONOO). Some substances, such as hydrogen peroxide (H 2 O 2 ), are said to be pro-oxidant, indicating that they lend themselves to the production of free radicals. Finally, there are substances called antioxidants that can scavenge free radicals and process them to either a less deleterious state or remove them altogether. While cells do require a certain redox balance in order to function, a shift in that balance toward an oxidant state indicates oxidant stress. This can occur due to an increase in free radical production, a decrease in antioxidants, or a combination of the two. When cells experience oxidant stress, they can be expected to show signs of oxidant damage to macromolecules including lipids, proteins, and nucleic acids (137, 207, 208, 235). In the case of lipids, the membrane bilayers are often vulnerable, leading to membrane lesions and loss of chemical and electrical gradients that can occur at both

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7 the cellular and organelle level (14, 37, 55, 88, 92, 124, 199, 206). Lesions in the sarcolemma can cause a loss of Na + K + and Ca 2+ homeostatic balance leading to a host of deleterious effects. In the case of mitochondria, lesions can lead to cytochrome C triggered apoptosis and loss of myonuclear number, as well as other harmful events. Protein oxidation and damage have received a great deal of attention in oxidative biology. The protein is acted on by a free radical and becomes oxidized resulting in either minor or major damage to the protein. In the case of minor damage, the protein can be repaired or refolded (17, 120, 174, 200). Severe damage results when the repair mechanisms are not sufficient to return the protein to a functional state. Should this occur, proteins are ubiquitinated and subsequently degraded by the 26S proteasome (101). Alternatively, recent evidence has suggested that degradation of oxidant-modified proteins can also occur by the 20S proteasome in a ubiquitin independent manner (51, 179). Oxidative attack of nucleic acids can occur at either the DNA level or the mRNA level (137). In the case of the former, modified bases may lead to errors during replication or transcription. Should the error be passed on during replication the result may be a non-functional gene in the daughter cell. This can lead to unchecked cell division and potentially cancerous growth. If free radicals attack mRNA the gene product will likely not be translated. Sources of Free Radicals There are several sources within the cell that can create free radicals. During oxidative phosphorylation, molecular oxygen is the final electron acceptor resulting in the production of two water molecules. Approximately 5% of oxygen; however, will result in the production of O 2 and free radical products rather than water (39). These free

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8 radicals will then begin to damage lipids, proteins, and nucleic acids. Xanthine oxidase acting on xanthine and hypoxanthine can also produce O 2 -. Superoxide is dismutated by superoxide dismutase (SOD) into the more stable H 2 O 2 While not a free radical itself, H 2 O 2 is very pro-oxidant, especially when exposed to Fe 2+ leading to a series of reactions termed Fenton chemistry (30). In short, these reactions involve iron interacting with several moieties to produce the free radical, OH. The free radical NO is used in the body for many things including as a neurotransmitter and a regulator of vascular tone (34, 44, 104, 231). It is produced in numerous tissues by several differing isoforms of NO synthase (NOS) including the neural isoform, the endothelial isoform, and the immune cell produced isoform. Despite its pluripotent biological roles, due to its unpaired valence electron, NO is also a free radical. When it interacts with O 2 the result is the cytotoxic ONOO, the second of the RNS (36, 182). Sources of Free Radicals During Immobilization During immobilization, there are several events that occur that lead to the production of free radicals. The first is an increase in the cytosolic Ca 2+ concentration, indicating a loss of Ca 2+ homeostasis and balance (96, 97, 206). Calcium activates proteases that catalyze the change from xanthine dehydrogenase to the free radical producing xanthine oxidase (149, 163, 214). Additionally, free iron also increases within the cell, permitting the formation of OH (30, 113, 114). Indeed, elevated OH levels have been measured in immobilized hind limbs (116). Alternative pathways leading to the production of free radicals may exist. When cytosolic Ca 2+ levels are high, the mitochondrial uniporter will begin to transport more Ca 2+ than it can expel resulting in elevated mitochondrial Ca 2+ concentrations (83, 98).

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9 As a result, the membrane potential is decreased and rate of electron transfer in the electron transport chain is reduced, potentially allowing a greater amount of electron leakage at complexes I and III and subsequent O 2 production (39, 58, 87). This is compounded by the fact that enzymes responsible for the removal of O 2 are reduced during disuse (115, 124). Finally, elevated Ca 2+ within the cell or mitochondria can increase the activity of NOS, leading to an increase in NO production (139). As mentioned earlier, NO can interact with O 2 and produce the highly cytotoxic ONOO. Further compounding matters is the fact that as more free radicals are produced, more Ca 2+ will be released from the SR, thus beginning a positive feedback loop where ROS propagates Ca 2+ release, while inhibiting Ca 2+ removal, and high intracellular Ca 2+ is facilitating ROS production (2, 79, 80, 194, 223). Regulation of Free Radicals In order to combat free radicals and oxidative stress, the body has several potent antioxidant enzymes and a host of other antioxidant molecules. As mentioned earlier, SOD catalyzes the formation of H 2 O 2 from O 2 by combining it with water. MnSOD is responsible for the dismutation of mitochondria mediated O 2 production, while CuZnSOD is responsible for cytosolic O 2 -. Hydrogen peroxide has itself been demonstrated to be harmful to the cell due to its instability; hence its removal is required. Glutathione peroxidase (GPX) decreases the activation energy of the reaction between glutathione (GSH) and H 2 O 2 resulting in the formation of water and oxidized glutathione (GSSH). Glutathione reductase can then reduce GSSH so that it can interact with H 2 O 2 once again. Catalase is also responsible for the breakdown of H 2 O 2 although it needs no other molecule to perform this role.

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10 There are other substances that are free radical scavengers that are not antioxidant enzymes. The first of these was alluded to above GSH. Two GSH can interact and scavenge one H 2 O 2 resulting in the formation of a glutathione dimer. Additionally, vitamins C and E have been demonstrated to be free radical scavengers. These compounds, along with others, interact to form a cascade of free radical removal such that some substances remove free radicals directly, while others, in effect, recharge scavengers (Figure 2-1). A breakdown in part of the chain can greatly attenuate the effectiveness of the antioxidants. Figure 2-1. Schematic of antioxidant scavenging system. In this example, Vitamin E scavenges the ROS, while either Vitamin C or GSH recharge Vitamin E. Further down stream, NADPH reduces GSSG, while lipoic acid reduces oxidized vitamin C (provided by Karyn Hamilton, personal communication). R Vitam i n E Vitam i n E GSH Vitam i n C GSSG Vitam i n C NADP+ NADPH DHLA LA R Skeletal Muscle and Unloading Models There are three common models used in research to induce atrophy. An obvious similarity among these models is that they reduce the normal load the muscle experiences and atrophy results (134). Two of these models are reduced use models, in which the muscle is still capable of contracting; however, the force the animal can generate has been greatly reduced. The third of these models involves transsection of the spinal cord, and, as such, is a true disuse model. As the purpose of this review is a discussion

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11 relevant to reloading, this review will focus only on models of reduced use, and accordingly, spinal transsection will not be addressed. Disuse atrophy and reduced use atrophy will be used interchangeably throughout. While this may technically be inaccurate, disuse atrophy has been used colloquially to encompass both reduced use and true disuse. The two models of reduced use atrophy are hind limb suspension (HLS; it is also commonly called hind limb unweighting, HLU) and hind limb immobilization (HLI). During HLS, the animal is suspended by the tail such that the hind limbs are no longer in contact with the cage bottom (7). The animal is generally given free reign over the cage and can move by way of the forelimbs. In this model the hind limbs muscles are capable of being recruited; however, they experience no load and produce no force. During HLI, the animals hind limbs are immobilized by cast fixation (22). Generally, immobilization occurs in the plantar flexed position causing shortening of the hind limb muscles and lengthening of the anterior limb muscles; however, some investigators have made use of a neutral position or a dorsiflexed position. Muscle groups immobilized in a shortened position experience significantly more atrophy than muscles immobilized in a neutral or lengthened position, which will experience either no loss of mass, or slight, but significant, hypertrophy (4, 26, 74, 99, 127, 171). During HLI, the animal is capable of recruiting fibers in immobilized muscles; however, muscle EMG activity is reduced by 85-90% of uncasted controls (90). Further, the force generated from a shortened muscle is not sufficient to prevent or even reduce atrophy when compared to a muscle of a HLS animal.

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12 Changes with Disuse Atrophy During disuse atrophy the muscle undergoes significant remodeling, yielding a smaller, less functional muscle. There are significant losses in mass and cross sectional area (10, 124). There is also a disproportionate loss in force causing specific tension to decrease, likely due to uncoupling of the excitation/contraction pathway (70). Mentioned earlier was a loss of Ca 2+ homeostatic containment and an elevation of intracellular Ca 2+ concentration (21, 96, 97, 206, 213). Further, the myosin heavy chain isoform should be expected to shift toward the faster isoforms. This is especially pronounced in the predominantly type I soleus. By shifting to faster isoforms, the muscle is better able to maintain power when force generation is falling, as power is a function of both force and contraction speed. During disuse atrophy there is also an increase in protein degradation rates and proteolytic components (52, 101, 125, 169, 198). While the precise mechanism of the elevated protein degradation is currently unknown, some aspects have been defined. It appears as though an early event in the pathway is the release of actin and myosin from the sarcomere likely through calpain cleavage at the Z-lines (91, 229). Once the contractile proteins are freed, they can then be ubiquitinated. The ubiquitination process involves several enzymes, classified as E1, E2, and E3, each performing a specific role within the cell. E1 is the ubiquitin activating enzyme and in an ATP dependant manner converts ubiquitin from an inactive form to an active form (47). E2 is a ubiquitin conjugating enzyme and carries activated ubiquitin to E3, ubiquitin ligase, that has already bound the damaged protein (128, 131, 132). E3 attaches ubiquitin to the damaged protein. Two isoforms of E3, atrogin (also called muscle atrophy F-box) and muscle ring finger-1

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13 (MuRF-1), are believed to be involved in degradation specific to atrophy as they are sharply elevated in several conditions causing atrophy (38, 75, 125, 152, 196, 198). Once a poly-ubiquitin tail, consisting of four or greater ubiquitin molecules, has been formed, the protein can be identified and degraded by the 26S proteasome (41, 227). The protein is denatured on the surface of the proteasome in an ATP dependant manner and subsequently broken down into polypeptides normally between 4 and 20 amino acids in length (23). Within the core of the proteasome are five catalytic sites capable of breaking peptide bonds including chymotrypsin-like activity (CT-L), trypsin-like activity (C-L), peptidyl-glutamyl-peptide hydrolyzing (PGPH), branch-chain amino acid preferring (BrAAP), and small neutral amino acid preferring (SNAAP) (175, 176). Each step, from calpain activity to 26S activity, has been demonstrated to be elevated during disuse atrophy. In addition to increased degradation, there is a very rapid reduction in protein synthesis occurring within hours of onset of disuse (121, 125, 225). There are three phases to protein synthesis including initiation, elongation, and termination and are all likely steps of regulation. It has been determined; however, that it is the elongation phase that is inhibited and causes the reduction in protein synthesis during disuse. In an elegantly designed study, Ku and Thomason (121) were able to determine differences between the influences of initiation, elongation, and termination and conclude that regulation of the elongation phase alone was responsible for compromised protein synthesis. It seems regulation of several key proteins involved in elongation may be to blame. Eukaryotic elongation factor 1A is responsible for the binding of tRNA to the ribosome. The demethylation of eEF-1A, by unknown mechanisms, will slow the

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14 elongation phase as tRNA will not be able to bind the ribosome (121). Eukarytotic elongation factor 2 is responsible for translocation. Phosphorylation of eEF-2, via eEF-2 kinase, causes a slowing in the translocation step. Interestingly, oxidative stress will cause an increase in the activity of eEF-2 kinase and a subsequent reduction in eEF-2 activity (180). Not only does eEF-2 kinase appear to be redox sensitive, but it also will increase activity in a Ca 2+ /calmodulin dependant manner as well (25). In a hind limb immobilized animal there is both an increased oxidative stress as well as an increased intracellular Ca 2+ concentration (96, 97, 116, 206). The summation of an increased protein degradation rate and depressed protein synthesis rate yields a sharp reduction in protein content within the cell (225). Protein content will continue to fall until a new steady state level is met at approximately three weeks when protein degradation is no longer elevated and protein synthesis has reached a new steady state. Oxidative Stress, Antioxidants and Unloading As demonstrated earlier, oxidative stress is clearly increased during periods of unloading. It seems that elevated free Ca 2+ leads to the production of O 2 via xanthine oxidase or mitochondria, which can lead to the production of other free radicals (Figure 2-1). These free radicals can then damage lipids, protein, and DNA. In the case of protein, the increased damage likely contributes to the increased protein degradation observed with disuse atrophy; thus, if oxidative damage is reduced, damaged protein content may be reduced, and, therefore, protein degradation rates may be reduced. Further, if oxidant stress could be reduced, elongation rates may be maintained. This leads to the hypothesis that supplementing with antioxidants during disuse atrophy may protect the muscle from atrophy and may help to maintain protein content.

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15 The antioxidant, vitamin E, has been utilized in several investigations with varying degrees of success. Kondo et al. (112) supplemented animals with vitamin E and reduced oxidative damage and increased muscle mass in HLI animals by approximately 20%, when compared to HLI alone. In addition, Appell et al. (10) found nearly 66% larger cross sectional area in vitamin E supplemented animals following HLI than with HLI alone. In another investigation; however, an antioxidant cocktail containing vitamin E failed to preserve muscle mass during HLS (107). Despite the noted variability in effectiveness, the rationale for using an antioxidant remains sound. Skeletal Muscle and Reloading Adaptations to Reloading As stated earlier, skeletal muscle is a highly plastic tissue and will respond to a change in workload by altering gene expression. In the case of reloading, skeletal muscle is transitioning from a state of disuse to a state of increased workload. This is accomplished by allowing the animal to re-ambulate, normally following hind limb unweighting or hind limb immobilization. In a matter of days, there is a dramatic increase in muscle mass, muscle cross sectional area, and force (140). Protein synthesis increases rapidly upon reloading with actin and cytochrome C synthesis peaking at four days (160). Protein degradation also increases dramatically during the initial stages of reloading. After only 18 h of reloading calpain, ubiquitin, and the 20S proteasome subunit mRNA are elevated (224). Additionally, both Ca 2+ independent and non-lysosomal protein degradation are elevated at 18 h (224). This continues up to one week later as the remodeling phase involves the removal of damaged protein prior to full recovery.

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16 Depending upon the duration of disuse and age of the animal, complete recovery may not occur with reloading. In adult animals, the muscle will atrophy and regrow as expected. In the case of old animals, the muscle will atrophy; however, it will only recover slightly (240, 241). Finally, in juvenile animals that have undergone an unloading/reloading cycle, muscles generally do not reach the size of muscles in control animals, even during adulthood (162). Pathways of Regrowth The mechanism of skeletal muscle regrowth appears to biphasic and fiber type specific; however, it is still highly debated and researched. There does appear to be consensus that during the first week of regrowth, the soleus regrows in a calcineurin-independent and satellite cell independent manner and an insulin-like growth factor-1 (IGF-1) dependent manner (20, 158). During the second week of regrowth it appears the soleus is dependent upon both calcineurin as well as satellite cells (158). The mechanisms of regrowth of the plantaris are less clear as evidence has been collected supporting both a calcineurin dependent and independent first week of growth, as well as an IGF-1 dependent first week of growth (20, 158). It does appear that the second week is calcineurin dependent and satellite cell independent (158). IGF-1 activates phosphatidyl-inositol 3-kinase (PI 3 K) (56), which in turn activates protein kinase B (PKB; Akt) through phosphoinositide-dependent kinase-1 and (PDK 1 and 2) (31, 59, 105). PKB activates three parallel pathways culminating in elevated protein synthesis. It has become clear in recent years that oxidant stress, heat shock, and potentially hsps 32 and 72 (likely not hsp 27) specifically, will cause an increased activation, i.e. increased phosphorylation, of IGF-1 pathway intermediates (56, 57, 64, 118, 202). Both oxidant stress and heat shock will increase PKB activation via PI 3 K,

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17 although some debate remains regarding an alternative heat shock/PKB activation pathway independent of PI 3 K and mitogen activated protein kinase activating protein-2 (MAPKAP-2) (117, 202). In addition, oxidant stress activation of PKB may be dependant upon intracellular Ca 2+ (50). These findings are of particular interest in a reloading model as there is both an elevated oxidant stress as well as an elevated Ca 2+ in reloading skeletal muscle fibers (discussed below). Reloading: Injury and Damage During reloading, the formation of sarcomeric lesions on skeletal muscle fibers is common and appears to increase in severity until approximately seven days of reloading, supporting elevated protein degradation data presented earlier (102, 226). These lesions are characterized by sarcomeric widening, myofibrilar loss, as well as myofibrilar misalignment, and are localized rather than dispersed (71, 135, 141, 151). Lesion formation is nearly instantaneous upon reloading and has been found to appear after only one lengthening contraction (226). Further, it appears that the initial step in lesion formation is damage to the sarcomere and perhaps more specifically, to myosin or titin (226, 242). It also appears that the soleus undergoes more severe lesion formation than the plantaris with the lesions occuring in 3-46% of fibers. Several reasons have been proposed to explain the disparity and variability, including differing recruitment patterns, cytoskeletal components of differing isoforms, and differences in contractile proteins and properties. To help clarify this issue, research has been conducted on reloaded muscle after exposure to microgravity in space (232). This study was able to discern several different fiber types within the same muscle including a slow phenotype as well as a hybrid phenotype expressing both type I and II myosin isoforms. Ninety percent of type I fibers

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18 had lesions on the sarcolemma, while only ten percent of hybrid fibers exhibited sarcolemmal lesions. While the authors considered a variety of causes for the difference in lesion formation between the two phenotypes, they cite difference in recruitment pattern as the most likely source. They argue that even within the same muscle, a type I fiber will be recruited more than a hybrid fiber. Accordingly, a type I fiber will be subjected to more stress and have a greater potential for damage. In addition to damage to the sarcolemma, the cytoskeleton also suffers damage during reloading. The cytoskeleton is comprised of a host of proteins and is responsible for transmitting force across the sarcolemma. This raises the possibility that, should the network be ruptured, force production may suffer. Dystrophin undergoes severe damage very soon after an eccentric contraction, primarily at the -COOH end (138). Meanwhile, associated cytoskeletal components such as spectrin and desmin are nearly unaffected (138). It has been shown that during reloading or pliometric injury, there is a loss of Ca 2+ homeostasis within the cell and a subsequent increase in Ca 2+ activated proteases, including calpain (5, 6). The -COOH end of dystrophin is particularly sensitive to calpain attack, which may help explain why dystrophin seems to be selectively damaged, while other cytoskeletal components are not (48, 89, 212). Reloading and the Immune Response As is common with many types of muscle injury, a reloading injury will result in activation of the immune system and recruitment of immune cells to the damaged region. The presence of free radicals within the cell can initiate a pathway, likely through p38 and NFB, leading to the production of IL-6 (119). Once produced, IL-6 can chemotactically recruit immune cells to the site of production, i.e., the damaged muscle fiber. Aiding in the chemotaxis of immune cells during reloading injury is the activation

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19 of both classical and alternative complement (69). Complement is a series of reactions that leads to the production of chemotactic moieties. Mitochondrial and cytoskeletal proteins in the extracellular space, likely seeping out through sarcolemmal lesions, will initiate the complement pathways. Further, complement inhibition will decrease immune cell infiltration (69). Despite the obvious infiltration of immune cells into fibers with injury resulting from reloading, questions have been raised regarding the true nature of their role. Earlier evidence has suggested they are deleterious particularly because they can generate a large amount of oxidant damage within the cell. Frenette et al. (70) have constructed a time course in which immune cell infiltration and the generation of tetanic force were superimposed on each other. They were able to show that following 10 d of unweighting and various durations of reloading, the greatest deficit in force came after only two hours of reloading, where an increase in immune cell presence did not occur until 24 h following reloading. This study indicates that the immune response is not responsible for reductions in force following reloading. Instead, given the short duration of force reduction, mechanical damage, oxidant damage, or E/C uncoupling is a far greater likelihood. Indeed, E/C uncoupling has been noted after only two hours of reloading (70). Additionally, both mechanical damage and/or oxidant damage could lead to Ca 2+ handling problems. Oxidant Damage During reloading the cell is subjected to a secondary oxidative stress, independent of the oxidative stress experienced during immobilization, akin to an ischemia/reperfusion type injury. Further, there is a loss of Ca2+ containment that occurs within the cell during reloading potentially due to damage to the sarcolemma or via Ca2+

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20 leak channels, although the precise mechanism is currently unknown (6, 96, 194). This loss of homeostatic balance likely contributes to the secondary oxidative stress that occurs during reloading in a similar fashion that occurs with unloading. The Ca2+ activated proteases, most likely calpain, convert xanthine dehydrogenase to xanthine oxidase and result in the production of O2(149, 163, 214). Similar to unloading, the iron catalyzed production of free radicals is also likely occurring (30). Additionally, the increased intracellular Ca 2+ concentration leads to an upregulation of Ca 2+ entering the mitochondria via the uniporter, resulting in a decreased membrane potential (83, 98). With a reduction in membrane potential comes a reduction in the rate of electron movement in the electron transport chain, increasing the possibility that electrons will leak from the electron transport chain and produce O 2 (39, 58, 87). Compounding matters is the reduction of MnSOD resulting from unloading, which is responsible for the dismutasing of O 2 (115, 124). Elevated intracellular Ca 2+ concentrations will also increase NOS activity and the production of NO, which can combine with O 2 to form ONOO (139). A positive feedback loop begins where increased Ca 2+ release propagates increased free radical production, and conversely, the increased free radicals both increase Ca 2+ release and inhibit Ca 2+ clearance (2, 79, 80, 194, 223). Regardless of source, the cell experiences this secondary bout of oxidant stress with a somewhat compromised antioxidant defense mechanism, making it potentially more potent and deleterious. While there were relatively few papers investigating oxidant stress and immobilization, there are fewer still investigating oxidant stress and reloading. In one investigation, reloading resulted in an increase in lipid oxidation as well as oxidized

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21 glutathione. In a reloaded group also given the antioxidant, vitamin E, there was less lipid oxidation and muscle mass was significantly larger when compared to reloaded controls (110). In another investigation, SOD supplementation attenuated the reduction in force associated with pliometric contractions both 10 min and three days after injury indicating that losses in force were not solely mechanical in nature, but also had a chemical or ROS mediated component (65, 242). Additionally, vitamin E supplementation has been demonstrated to be of benefit as it has reduced creatine kinase release and reduced oxidative damage (110, 148, 157). In contrast, other studies have shown that vitamin E supplementation did not maintain torque, prevent Z-line streaming, reduce creatine kinase release, or reduce macrophage infiltration following eccentric damage nor was it beneficial to downhill running rats (15, 233). Heat Shock Proteins Overview When the cell encounters a variety of stressful conditions a group of proteins called stress shock proteins can be differentially expressed. Among the first characterized were the heat shock proteins (hsp), which were first discovered to respond to a heat stress. More recently, other stimulators have been identified including oxidative damage, exercise, pharmacological agents, and UV light (for rev.159). The hsps can be divided into five families including hsp 90, 70, 60, 40, and the small hsps (shsp). These proteins function to prevent protein degradation as well as assist in the elimination of proteins that are too badly damaged for repair (236). The hsp 70 family member, hsp 72, and the shsp family members, hsp 27 (hsp 27 in human is analogous to 25 in mouse and 28 in rats) and hsp 32 (also commonly called Heme Oxygenase-1; HO-1) are of particular interest when

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22 considering reloading as they are heat inducible and serve a variety of functions that may be beneficial to a reloading muscle fiber. Heat Shock Proteins 27, 32, and 72 Hsp 27 is by far the most common shsp with estimates as high as 3.4-ug/mg protein in human biceps (220), while hsp 32 and 72 are readily inducible. Collectively, hsp 27, 32, and 72 have been shown to refold damaged or nascent proteins (chaperone) (16, 60), interact with cytoskeletal components (181), help reduce oxidant stress (95, 109, 217), and prevent apoptosis (16, 24, 73, 154, 155). In its oligomeric form, hsp 27 functions to act as a chaperone in an ATP independent manner. While in this conformation it can remove heat damaged proteins, reduce protein aggregation, refold damaged proteins and can directly reduce ROS, thereby reducing the potential for oxidative damage (193). Phosphorylation, occurring at one of three serine residues, is one of the primary means of regulating hsp 27 activity (94). The MAPK, p38, initiates a cascade that goes through MAPKAP 2/3 and culminates with the phosphorylation of hsp 27 (82, 153). Upon phosphorylation, the activated monomer will dissociate from the large oligomer and complete its job within the cell (103, 193). As conditions become more stressful, hsp 27 becomes progressively more phosphorylated (94). In its tri-phosphate form, hsp 27 exists in high levels as rod-like tetramers (193). Currently, it is unknown what effect phosphorylation at the various sites has on the role of this protein. Further, the precise role each of the conformations this protein can take is also yet to be determined. While hsp 27 is activated through a MAPK signaling pathway, hsp 72 is activated by a more complex series of events. Under control conditions, hsp 72 is bound to heat shock factor 1 (hsf-1) forming a dimer. Upon stress conditions, such as increased

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23 intracellular protein damage, hsp 72 dissociates from hsf-1 and binds damaged proteins (18). Hsf-1 then trimerizes and migrates to the nucleus where it binds the heat shock element in the promoter region of the hsp 72 gene and facilitates transcription. Following the repair of damaged proteins, hsp 72 is free in the cell and will once again bind hsf-1, decreasing its ability to trimerize and produce more hsp 72 (13, 136). Alternatively, in the heart, it appears as though stretch activated ion channels can lead to the activation of hsf-1 and the subsequent increase in hsp 72 content (40). Further evidence of the differing activation pathways of these two proteins is gained when glucocorticoids are administered. Knowltons group was able to demonstrate that hsp 72 was elevated along with hsf-1, while hsp 27 remained at baseline levels (221). Far more is known about hsp 72 in terms of function within the cell. Expanding the role of hsp 72 already described is an involvement in protein synthesis and degradation. This protein has been shown to modulate the rate of the elongation process, thereby affecting protein synthesis (16, 122). It can also reduce or prevent protein aggregation and appears likely that it can facilitate recognition of damaged proteins within aggregates by partially unfolding them to allow ubiquitination (29, 129, 130, 204). Large protein aggregates can inhibit proteasome activity and create a potentially cytotoxic accumulation of damaged proteins (81). By reducing these aggregates, proteolytic degradation can be maintained. Through a yet to be described mechanism, hsp 72 also increased MnSOD and Bcl-2 levels within an I/R heart, which may provide an additional mechanism of heat shock protein induced cardio-protection (222). Hsp 32 is activated in a similar fashion as hsp 72. A heat shock element is found in the promoter region of the rat hsp 32 gene (184). In addition, there are binding sites

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24 for NFB as well as activating protein-1 (AP-1), which is redox sensitive (123, 185). In addition, IL-6 as well as a reduced GSH content will increase hsp 32 expression (191, 195). Furthermore, free heme and heat will induce an increase in hsp 32 expression (62, 63, 143, 184). Once induced, hsp 32 will degrade heme into three products including CO, biliverdin, and iron. CO can have anti-inflammatory effects as it regulates macrophage infiltration and cytokine release (28, 177). Further, CO can activate p38, which will, in turn, activate a host of other protein kinases responsible for, among other things, the activation of hsp 27 (24). Finally, CO has repeatedly been demonstrated to reduce apoptosis (24). Biliverdin is a water-soluble antioxidant that is quickly converted into the hydrophobic antioxidant, bilirubin. The final product of hsp 32 activity is perhaps the most complicated. The release of free iron within the cell raises the possibility that it will increase free radical synthesis and hence, oxidant stress, as Fe 2+ can participate in the Fenton reaction. Upon release of iron into the cytosol, iron regulatory protein, which blocks ferritin translation as it is bound to the mRNA, dissociates and ferritin is increased within the cell (61). Ferritin binds free iron, making the elevation of free iron only transient. Further, when iron is bound to ferritin in an oxidizing environment, such as during disuse or reloading, O2can attack ferritin and cause the transient release of iron as the pro-oxidant Fe 2+ (11, 195). Despite the obvious risk of a transient increase of free iron within the cell, the summation of these events is a reduction of free iron due to the increased ferritin (161, 178, 195). Heat Shock Proteins, Oxidant Stress, and Potential as a Reloading Intervention Among the many roles hsps can perform within the cell, their antioxidant properties may be of most importance to a reloading skeletal muscle. Elevations in hsp 27 have been demonstrated to provide protection against H 2 O 2 as well as oxidant damage caused

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25 by TNF via NFB (238). In rats with simulated Huntingtons Disease, hsp 27 was associated with higher survival rates and lower ROS production despite no reductions in the expression of the Huntington protein, the cause ROS production (154, 237). Another potent mode of cellular protection in the face of oxidant stress conferred by hsp 27 is the reduction of GSSH to GSH (170). The antioxidant effects of hsp 32 are primarily conferred through the release of biliverdin and bilirubin from the degradation of heme, although the effects of ferritin are also important in maintaining redox balance. Biliverdin and bilirubin have been found to be as effective an antioxidant as vitamin E (217). It seems that they function through a variety of means to reduce oxidant stress including acting as a peroxyl radical trap, breaking the oxidant chain reaction in membranes, protecting against OH attack, and decreasing O 2 (166, 216). For example, both hsp 32 induction as well as bilirubin supplementation were quite effective in protecting cells and reducing oxidant damage following an ischemia/reoxygenation event (68). Further, infarct area and mitochondrial damage were reduced in ischemia/reperfused hearts following either hsp 32 induction or bilirubin exposure (45). In addition, free heme is pro-oxidant which indicates that its removal from the cell is paramount. In a similar fashion, hsp 72 has been shown to increase tolerance to oxidative stress. Muscles from transgenic mice over-expressing hsp 72 showed higher force generation following hypoxic fatigue, which the authors attributed to the protective effect of hsp 72 to ROS damage (205). Further, cells transfected with an hsp 72 over-expression gene demonstrated less cell death and damage when exposed to H 2 O 2 (109). More common; however, is an association between hsp 72 and a protective effect against

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26 oxidant stress. It is worth noting that in many of these studies, hsp 27 and 32 were not assessed and likely provided some sort of protective effect as well. Untrained animals that are forced to exercise demonstrate higher oxidant stress, as their antioxidant enzymes appear to be overwhelmed. Concomitantly, hsp 72 is over-expressed in these animals following exercise, while their trained counterparts have enzyme activity levels capable of handling the oxidant stress of one exercise bout and show no elevation in hsp 72 expression. Heat stress has been shown to be protective against an array of oxidative insults including skeletal muscle H 2 O 2 exposure, skeletal muscle ischemic injury, and liver carbon tetrachloride exposure (72, 165, 239). As has been demonstrated previously, there is an increase in oxidant stress during immobilization. The literature is inconsistent as it relates to changes in hsp 72 expression with this type of intervention. It does appear though, that female rats demonstrate a decline in expression, while the expression in male rats does not change (164, 201). Regardless, there is not an increase in expression as may be expected during such a stress. Taking advantage of what appears to be a biological oversight, two groups have successfully used a heat intervention to attenuate muscle loss due to disuse atrophy (164, 201). In addition, oxidant stress was reduced in the group that was both immobilized and heated when compared to one that was only immobilized (201). Given that there is also a large increase in oxidant stress during reloading, it stands to reason then, that providing a heat stress will likely result in elevated hsp expression as well as decreased oxidant damage. As mentioned earlier, vitamin E supplementation has been successfully used to reduce oxidant damage and enhance muscle regrowth following immobilization (110). Further supporting the role of hsp s in reloading skeletal muscle

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27 are data that show that age matched transgenic mice over-expressing hsp 72 recovered from a bout of pliometric contractions faster than wild-type adult and old mice (147). These mice also did not suffer the well-characterized secondary drop in force production occurring several days after injury. Further, adult transgenics also had a smaller damaged muscle area than wild-type controls. The summation of this body of evidence leads to the hypothesis that following immobilization, heating will decrease oxidant damage to proteins and lipids during reloading. Furthermore, it is anticipated that heat stress during reloading will result in enhanced hypertrophy and higher IGF-1 pathway intermediate activation during regrowth.

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CHAPTER 3 METHODS Design All procedures and experiments were conducted with the approval of the Institutional Animal Care and Use Committee at the University of Florida under the animal use protocol designated D736. Animals were housed in a 12 hr light/dark photoperiod in an environmentally controlled room. Upon arrival in the facility, animals were handled daily for one week prior to the initiation of experiments in an effort to minimize contact stress. Male Sprague-Dawley rats were randomly be divided into six groups including a control group (Con) (n=10), a heated control group that will receive multiple heat treatments on alternating days (ConH) (n=6), a group that is immobilized for seven days (Im) (n=10), a group that is immobilized for seven days and receives a heat treatment 24 hours before sacrifice (ImH) (n=6), a group immobilized for seven days and allowed to reload for seven (R7C) (n=10), and a group immobilized for seven days and allowed to reload for seven that receive heat treatments on alternating days during reloading (R7H) (n=10; Figure 3-1). Animals given heat therapy and immobilization will be heated 24 hours prior to cast removal in order to assure that hsps are elevated when the casts are removed. Animals assigned to the R7C and R7H will be allowed to reload for seven days before sacrifice (Figure 3-2). 28

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29 True Control (Con) Heated Control HCon Heated like R7H Control Not Heated (Im) Reloaded 0 days Heated (ImH) Reloaded 0 days Not Heated (R7C) Reloaded 7 days Heated (R7H) Reloaded 7 days Immobilized 7 days Reloaded Sprague-Dawley Male 320 g Figure 3-1. Schematic of study design. Animal s will be randomly assigned to one of six groups. Control animals will be treated id entically to other groups except that they will not be immobilized. Animals not receiving a heat treatment will be anesthetized identically to heated anim als; however, the core temperature will only be maintained rather than elevated to 41-41.5 C as in heated animals. Figure 3-2. Schematic of protocol for one hypothetical week. Anim als will be treated such that representatives from multiple groups will be collected at the same time. This potentates the probability that groups will be trea ted identically. General Procedures Immobilization and Reloading Anesthesia was induced with a 5% isof lurane gas oxygen mixture and maintained with a 1.5-2% isoflurane gas oxygen mixture administered through a calibrated air flow meter (Veterinary Equipment and Technical Se rvice, Gainesville, FL). Animals were

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30 then immobilized bilaterally in the plantarflexed position as to cause maximal atrophy in the triceps surae muscle group in accordance with the model described by Booth and Kelso with modifications (22). Briefly, animals were wrapped in a protective adhesive (Medipore Dress-it, 3M, St. Paul, Minnesota) so that the animals would not come in contact with the plaster. The wrap begins in the supra-abdominal area, below the level of the ribs, and continues down the abdomen of the animal and stops in the infra-abdominal area and continues down the hind limbs. A quick drying plaster was then applied and allowed to dry (Specialist, Johnson and Johnson, New Brunswick, New Jersey). Finally a Plexiglas wrap was applied so that rats could not chew through the cast (Scotchcast Plus, 3M, St. Paul, Minnesota). In order to remove the casts, the animals were again anesthetized with isoflurane. A rotor with a cutting wheel is used to remove the Plexiglas wrap. The casts were then softened with warm water to make the plaster easier to cut. The cutting wheel was then used again to remove the plaster cast. Animals were sacrificed or placed back into their cages and allowed to return to normal activity for a period of seven days depending on which group they were assigned. Heat Treatment Animals were anesthetized using isoflurane, as detailed above. A rectal probe was inserted and secured to the tail to ensure that it would not become displaced (YSI, Yellow Springs, OH). The animal was then wrapped in a pre-warmed thermal blanket (Kaz, Hudson, NY) such that the tail and head were exposed. The tail was left exposed because it serves as the anchor for the rectal probe; hence it cannot be curled into the blanket. The head was visible enough to ensure that the nose cone was secure so that accidental recovery from anesthesia would not occur.

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31 Core temperature was continuously monitored and recorded every two minutes. Heating time began as soon as the core temperature of the animal breached 41C and temperature was maintained at 41-41.5C for 30 min. At 30 min, the animals core temperature was lowered via convection cooling and continually monitored until the core temperature was below 39.9C. Animals not receiving a heat treatment were treated identically, except that core temperature was maintained for the 30 min period. Animals receiving a heat treatment were heated 24 h prior to cast removal. We have found that hsp expression peaks between 24 and 48 hrs following heating. Animals allowed to reload for seven days will be heated 24 h prior to reloading and every other day following until sacrifice. Muscle Removal and Sample Preparation On the day of sacrifice, a surgical plane of anesthesia was induced via interperitonial pentobarbital injection. Then, the soleus was removed, trimmed of excess fat, tendon, and nerve, blotted, weighed and immediately frozen in liquid nitrogen chilled isopentane for subsequent analysis. To determine if muscle water content is altered, total water content of muscles was determined by using a freeze drying technique incorporating a vacuum pump with a negative pressure of ~1 mm Hg. The measurement was terminated when the same weight was recorded three times in succession during 48 hr interval. Homogenization occurred following the technique of Solaro et al. (209). Briefly, mass to buffer ratio was 20:1 such that if 0.03 g frozen tissue were utilized, it would require 0.6 ml buffer. The resulting homogenate was then centrifuged at 300 g for 10 min to remove cellular debris and the supernatant removed while the pellet was

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32 discarded. Protein concentration was then determined using the biuret technique of Watters (234). Biochemical Procedures Western Blot Samples were diluted to 1 mg/ml in sample buffer containing 62.5 mM Tris (pH=6.8), 1.0% SDS, 0.01% bromophenol blue, 15.0% glycerol, and 5% -mercaptoethanol. Samples were denatured via heating to 60C for 15 min in a glass bead heater. Precisely 15-ug protein is loaded into 4-20% vertical precast gels (Cambrex, Rockland, ME). Samples were then electrophoresed at room temperature for 30 minutes at 50 v followed by 90 min at 120 v (BioRad, Hercules, CA). Gels were removed from the electrophoresis apparatus and allowed to condition for 15 min in transfer buffer containing 25 mM Tris, 192 mM Glycine, 0.02% SDS, and 20% methanol (pH=8.3). Following the conditioning period, horizontal electrophoresis (100 v, 60 min, 4C) was performed such that proteins were transferred to a nitrocellulose membrane with a pore diameter of 0.2 um (BioRad, Hercules, CA). Alternatively, a dot blot was performed such that15 ug of sample was added directly to the membrane and proceeds as a normal western blot. Membranes were then washed in Tris-buffered saline containing .1% Tween 20 (TTBS). Membranes were blocked by exposure to a 5% dehydrated milk TTBS solution for 60 min. Membranes were washed for ten minutes, three times and exposed to the appropriate primary antibody as follows: hsp 25 (SPA 801, Stressgen, Victoria, British Columbia), hsp 72 (SPA 810, Stressgen), 4-hydroxy-2-nonenol (HNE; HNE11-S, Alpha Diagnostic International, San Antonia, Texas), nitrotyrosine (NT; #9691, Cell Signaling Technology, Beverly, Massachusetts), AKT (#9272, CST), phospho-AKT (#9271, CST), GSK3 (#9332, CST), phospho-GSK (#9331, CST),

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33 p70S6K (#9202, CST), and phospho-p70S6K (#9205, CST) for 90 min. They were then washed three times, for ten minutes and exposed to the appropriate secondary antibody for 60 min (Amersham). The secondary antibody was diluted in TTBS containing 1.5-2% milk protein. Membranes were then washed for ten min, three times and exposed to ECL (Amersham, Little Chalfont, Buckinghamshire, England) for two min. Finally, the membranes were placed in a Kodak Image Station 440 CF developer and the emitted signal captured. The signal was analyzed using the Kodak ID Image Analysis Software (Eastman Kodak Scientific Imaging Systems, Rochester, NY). Glutathione Peroxidase All antioxidant enzymatic assays will be performed in triplicate in microplates using a Spectramax 190 microplate reader (Molecular Devices, Downingtown, Pennsylvania) using homogenate further diluted to 1:100 in PBS buffer. GPX activity was determined by the method of Flohe and Gunzler (67) and was based on the change in NADPH absorbance from the reaction catalyzed by GR. 240 ul reaction cocktail containing .2974 U/ml GR, 1.25 mM GSH, and .1875 mM NADPH was added to a microplate well followed by 30 ul homogenate. Following a three minute incubation at 25C, 30 ul t-butyl hydroperoxide was added to the plate. The plate was read every 15 sec for five minutes at 340 nm. A blank was run and will substitute the homogenate for PBS. Activity was determined by first identifying the linear portion of the resulting graph. Change/minute will be divided by 6.22 (extinction co-efficient for NADPH) and multiplying by 2 (2 GSH:1 NADPH). Activity was then multiplied by the dilution factor and blank activity determined and subtracted from the total activity.

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34 Glutathione Reductase Glutathione reductase activity was determined by the method of Carlberg and Mannervik (35). Briefly, a reaction cocktail was first prepared containing three solutions. The first was 0.2 M PBS solution containing 2 mM EDTA. The second was 2mM NADPH in 10 mM Tris-HCl (ph 7.0). The final solution comprising the reaction cocktail was distilled water. The three were combined such that the mixture is 58.8% EDTA solution, 5.9% NADPH solution and 35.3% water. They were mixed and 170 ul is added to each well. Next, 20 ul sample was added to each well. The two were allowed to incubate for 2 min at 30C and then 10 ul water (blanks) or GSSH solution (20 mM) was added to the wells. Plates were read every 15 sec for three minutes at 340 nm. Activity was calculated by subtracting the blank change/minute from the change/minute recorded from each sample. Like before, it was divided by 6.22 to account for the extinction co-efficient of NADPH and multiplied by 2 to account for the GSH:GSSH ratio. Finally, this value was multiplied by the dilution factor. Catalase Catalase activity was determined by the method of Aebi (3). This assay is based on changes in absorbance due to the degradation of H 2 O 2 Sample was prepared as before with the addition of several extra steps. Following centrifugation, ethanol was added in a 1:10 v/v ratio and incubated for 30 min. Next, 1% triton was added in 1:10 v/v ratio and incubated for 15 min. This assay was run on plates designed for readings in the UV range. Additionally, only one column was run at a time due to the rapid reaction rate. Thirty-five microliters sample was loaded into each well. Next, 255 ul of H 2 O 2 solution containing,10 mM in 100 mM PBS (buffer for blanks), was then loaded into each well. The reaction was immediately read for one minute with data being recorded every 5

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35 seconds. Due to the abnormal kinetics of the catalase reaction, there is no defined unit for catalase activity. Instead, activity was determined using a first order reaction rate constant. The calculation was given by the equation: K= (2.3/t) [log10 ((initial absorbance blank)/(final absorbance blank)) Activity = K*dilution factor Superoxide Dismutase Mn super-oxide dismutase (SOD), and CuZnSOD activity was determined simultaneously by the method of McCord and Fridovich (150). Cytochrome C was reduced by O2-. The reduction of cytochrome C was slowed by the addition of SOD. To perform the assay, 180 ul reaction cocktail containing 16.6mM purine and 88.8 uM cytochrome c in 100mM PBS was added to each mircoplate well. Next, 15 ul homogenate or buffer (blanks) is added followed by 20 ul KCn or buffer. The addition of KCn will cause CuZnSOD not to function. Finally, 60 ul xanthine oxidase solution was added containing 0.2% xanthine oxidase in PBS. Readings were taken every 15 sec for four minutes at 550 nm. Activity was determined by the following calculation: U/gww = [(blank change/minute homogenate change/minute)/(0.5 blank change/minute)]* dilution factor CuZnSOD activity = Total SOD MnSOD (with KCn). Ferrous Oxidation Xylenol Orange In order to determine the ferrous oxidation of xylenol orange (FOX), muscles must be homogenized in methanol at a dilution of 1:10 in accordance with Hermes-Lima et al. (86). Samples were homogenized in cold methanol at a 1:10 w/v ratio. Homogenates were centrifuged at 1500 rpm for 10 min, and the supernated removed. The reaction cocktail containing 75 ul 1 mM FeSO4, 30 ul .25 M H2SO4, and 60 ul 1 mM Xylenol

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36 Orange was prepared and added to the mircoplate well. Next, 129 ul water was added, followed by 6 ul sample. These will be allowed to incubate for 24 h in the dark. They will then be read in triplicate at 580 nm. The results will be plotted against a standard curve constructed from cumene hydroperoxide. Total hyroperides are calculated as follows: mmol hydroperoxide/gr wet weight = (mmol concentration dilution factor)/grams tissue. ELISAS Protein carbonyls were determined from the Zentech PC Test (Zenith Technology Corp Ltd, Dunedin, NZ) (32). Hsp 32 content was measured using the Rat HO-1 ELISA Kit (EKS 810, Stressgen). Statistical Analyses Data was compared using a one-way ANOVA in a predetermined comparison involving the Con, Im, R7C, and R7H. Further, the ImH group will be used to show hsp levels upon removal of the casts in animals that were heated and immobilized in comparison to animals in the Im group. Finally, the ConH groups will be used to show the effects of the heating protocol on hsp expression. ANOVAS that result in an F-test that is significantly different will be compared using a Newman-Keuls post hoc test. Alpha will be set at p<0.05.

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CHAPTER 4 RESULTS As mentioned earlier, two control groups were included in addition to the true control. The first of these is identified as ConH and was treated like the Con except that it was heated in a similar fashion as the R7H group. It was included to show that our heating protocol would increase hsps and help to identify any variable that may respond to our heating treatment. The second of the additional controls is the ImH group. This group was treated like the R7H group, except that it was killed immediately prior to reloading. The ImH group is included to determine if hsps are elevated upon reloading as anticipated. Except where indicated, there was no difference between the Con and the ConH and the Im and the ImH groups. The presentation of the results begins with descriptive data from the whole animal and the intact muscle, followed by indicators of oxidant stress and antioxidant enzyme activities, culminating with data that may help to explain potential mechanism behind these observations. Whole Body Measures Initial body mass did not differ between groups (Table 4-1). One week of immobilization resulted in an approximate 10% weight loss for all immobilized groups. During that same time period, the Con group increased body mass by approximately 7%. Animals in the R7C and R7H groups were then allowed to reambulate for one week. While this period did prevent further reductions in body mass, it did not result in an 37

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38 increase in body mass. During the same time period, Con continued to increase body mass by another 6%. Table 4-1. Body weight changes at various time periods during this investigation. Values are shown in grams and are presented as means SEM. Con Im R7C R7H Initial Body Mass (g) 3573 3573 3515 3584 One Week Body Mass (g) 381 318 312 317 Two Week Body Mass (g) 405 NA 314 301 All immobilized animals were match fed during this experiment. During the unloading phase, there was no distinction in treatment between groups and as such, ad libitum food consumption is similar across groups at 16 g/day. Beginning 24 hr prior to reloading (day of first heating) animal feeding was matched to the R7H group. This match was successful as both the R7C and the R7H ate approximately 15 g of food daily for the week of reloading. Con rats were allowed to eat ad libitum for the duration of the experiment and ate approximately 23 g of food daily. Animals were allowed to drink water ad libitum during this experiment. Rats in the Con group drank approximately 42 g water daily, which was significantly higher than all other groups. During the immobilization phase of the experiment, animals drank approximately 30 g water daily. This did not differ statistically from the 33 g water consumed during the reloading phase of the experiment in either the R7C or the R7H groups. Whole Muscle Measures Upon sacrifice of each animal, soleus wet weight was determined immediately prior to freezing (Figure 4-1). Immobilization resulted in a 35% reduction in muscle mass when compared to Con. While one week of reloading appeared to increase mass when compared to the immobilized muscles, the increase of nearly 20% failed to reach

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39 statistical significance. When reloading was combined with heat, the result was a significantly larger muscle when compared to the muscles of the Im group. Furthermore, the addition of heat to reloading caused significantly greater hypertrophy when compared to reloading alone. Muscle water content was also determined and did not differ between groups, indicating edema was not present in reloaded muscles at the time of sacrifice (Table 4-2). Con Im R7Con R7H 0.0 0.1 0.2 grams Figure 4-1. Wet muscle mass in control animals (Con), following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H). Data is presented as means SEM. represents different from Con; represents different from R7H. When muscle mass was made relative to body mass (mg muscle mass/g body mass), a similar pattern emerged (Table 4-2). Immobilization resulted in significant atrophy when compared to Con. Reloading; however, returned this measure to Con values. Heating in conjunction with reloading further augmented this measure, as it was significantly greater than reloading alone and did not differ from Con.

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40 Table 4-2. Relative muscle mass, water content, and wet weight to dry weight ratio following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H). Data is presented as means SEM. represents different from Con; represents different from R7H. Con Im R7C R7H Relative Muscle Mass (mg/g) 0.44.01 0.35.01 0.41.02 0.48.03 % Water 78.2.5 77.5.4 78.1.5 78.5.7 Wet:Dry Ratio 4.61.12 4.45.08 4.59.10 4.70.15 Oxidative Damage Oxidative damage was assessed in lipids by measurement of both total lipid hydroperoxides (FOX) and lipid oxidation end products (HNE). Total lipid hydroperoxides did not differ between groups; however the relative content of lipid oxidation end products did differ between groups (Figure 4-2). In this measure, immobilization increased HNE products when compared to Con. Reloading further increased HNE products, as the R7C group was significantly higher than Con and Im. Heating in conjunction with reloading returned HNE products to Con values. In regard to protein oxidation, measures of toxic aldehyde and ketone attack (protein carbonyls) as well as nitrosylated tyrosine residues (NT) were made. Protein carbonyls did not differ between groups indicating that toxic aldehydes and/or ketones may not be a significant cause of injury in this model at these time points. However, the results of the NT dot blot assay were similar to the pattern of HNE change. Immobilization resulted in a significant increase in damage that was still present during reloading. Reloading in combination with heat eliminated this damage as NT was not different between the R7H group and the Con group (Figure 4-3). These data were confirmed by the conventional NT western blot, which revealed similar findings (Figure 4-3).

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41 Con Im R7C R7H 0.0 2.5 5.0 7.5 10.0 # Arbitrary OD Units Figure 4-2. 4-hydroxy-non-enol as determined by immuno-blotting following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H). Data is presented as means SEM. represents different from Con; represents different from R7H; # represents different from R7C. Con Im R7C R7H 0 1 2 3 4 5 6 7 Arbitrary OD Units Figure 4-3. Nitrotyrosine as determined by dot blot immuno-blotting (top) and conventional western blotting (bottom) following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H). Data is presented as means SEM. represents different from Con; represents different from R7H.

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42 Con Im R7C R7H 0.0 0.5 1.0 1.5 2.0 2.5 Arbitrary OD Units Figure 4-3. Continued Anti-Oxidant Enzymes As it is clear that the heat intervention does provide the cell with increased resistance to oxidant damage it follows that this effect may be due to changes in antioxidant enzyme activity. In this regard, both CuZn super oxide dismutase (SOD) and MnSOD activities were measured along with catalase (Cat), and the glutathione handling enzymes glutathione peroxidase (GPX) and glutathione reductase (GRX) (Table 4-3). MnSOD, GPX, and GRX activities did not differ between groups. Immobilization caused an increase in CuZnSOD activity, which was maintained during reloading. Reloading combined with heat; however, lowered this measure to Con levels. Catalase activity was significantly elevated with immobilization. Reloading lowered catalase activity; however it was still elevated over Con values. Reloading with heat lowered catalase activity to Con levels. These data suggest that heating does not increase antioxidant protection by increasing antioxidant enzyme activity as in no instance was R7H enzyme activity increased above that of R7C.

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43 Table 4-3. Activities of various antioxidant enzymes following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H). Data is presented as means SEM. represents different from Con; represents different from R7H; # represents different from R7C. Con Im R7C R7H MnSOD (U/gww/min) 356 304 261 288 CuZnSOD (U/gww/min) 212 310 293 229 Catalase (U/gww/min) 2.20.07 2.67.04# 2.44.09 2.30.06 GPX (U/gww/min) 7.20.43 6.77.37 8.04.41 6.96.33 GRX (U/gww/min) 1159 1137 1328 1138 IGF-1 Pathway Activation From the data above, it appears as though heating during reloading enhances muscle regrowth following one week of immobilization. In order to determine if this augmentation is due to IGF-1 pathway mediated protein synthesis, the protein content and activation state of Akt and its downstream targets, Gsk and p70s6k, were determined. Akt content was significantly reduced following one week of immobilization when compared to all other groups (Figure 4-4). Reloading; however, resulted in a significant elevation in Akt content when compared to all other groups. Interestingly, in conjunction with heat, reloading returned Akt content to a level that was similar to Con. In a similar fashion, Akt activation (as measured by Akt phoshorylation) was reduced following one week of immobilization. Both reloading and reloading with heat resulted in a two fold increase in Akt activation compared to Con. Since Akt content was higher in R7C than R7H group, and pAkt was similar in both groups, it suggests that a greater proportion of Akt is phosphorylated in the R7H group, which could indicate greater pathway activation in this group. In the ConH group, heating resulted in a significant reduction in Akt content as levels fell to approximately 35% of Con. Surprisingly, this reduction was not found

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44 when phospho Akt was assessed. This could further substantiate the notion that heating increases IGF-1 pathway activation Con Im R7C R7H 0 1 2 # *#*Arbitrary OD Units Con Im R7C R7H 0.0 0.5 1.0 1.5 2.0 2.5 #**Arbitrary OD Units Figure 4-4. Akt content (top) and phosphor-Akt content (bottom) as determined by immuno-blotting following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H). Data is presented as means SEM. represents different from Con; represents different from R7H; # indicates different from R7C; indicates different from Im. Immobilization did not reduce Gsk content nor change Gsk phosphorylation when compared to Con (Figure 4-5). Reloading significantly increased Gsk content in the R7C and R7H groups. Heat during reloading caused a significant increase in Gsk

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45 phosphorylation when compared to all other groups, while reloading alone did not increase Gsk phosphorylation state. Because reloading alone failed to increase Gsk phosphorylation a breakdown in IGF-1 pathway signaling may be occurring. The lack of change in phospho Gsk would decrease protein synthesis because a greater amount of Gsk is present in the muscle in its active state. By comparison, in the R7H group the increase in Gsk content is met by an increase in the phophorylation state meaning that even though Gsk content is increased, it is being inhibited at a greater proportion than in the R7C group, indicating a preservation of IGF-1 pathway signaling. Con Im R7C R7H 0.0 0.5 1.0 1.5 2.0 2.5#**Arbitrary OD Units Figure 4-5. Total Gsk (top) and phosphorylated Gsk (bottom) content as determined by immuno-blotting following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H). Data is presented as means SEM. represents different from Con; represents different from R7H; # indicates different from R7C; indicates different from Im.

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46 Con Im R7C R7H 0.0 0.5 1.0 1.5 2.0 2.5#*Arbitrary OD Units Figure 4-5. Continued P70s6k, a protein synthesis promoter, is downstream of Akt in the IGF-1 pathway and is activated in response to an increase in Akt activation. One week of immobilization failed to reduce p70s6k content; however, one week of reloading caused a significant elevation in the expression of this protein when compared to both Con and Im, regardless of heat (Figure 4-6). Furthermore, immobilization resulted in a significant reduction in p70s6k activation. Like total protein, reloading and reloading with heat resulted in a significant increase in activation of this protein. Additionally, heating resulted in significant reductions in p70s6k content in the ConH group when compared to the Con group and in the ImH group when compared to the Im group. Surprisingly, like Akt, the reduction in p70s6k content did not affect phosphorylated p70s6k content in either the ConH or the ImH groups as both were similar to the Con and Im groups, respectively.

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47 Con Im R7C R7H 0.0 0.5 1.0 1.5 2.0 2.5#**Arbitrary OD Units Con Im R7C R7H 0 1 2#**Arbitrary OD Units Figure 4-6. Total p70s6k (top) and phosphorylated p70s6k (bottom) content as determined by immuno-blotting following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H). Data is presented as means SEM. represents different from Con; represents different from R7H; # indicates different from R7C; indicates different from Im. Heat Shock Proteins As the antioxidant enzymes did not cause the decrease in oxidant damage found in this investigation, it is reasonable to suggest that increases in hsps may have played a role. Hsp 27 was significantly reduced following immobilization when compared to all other groups (Figure 4-7). Following one week of reloading, hsp 27 levels increased to above Con levels. Application of a heat treatment further elevated hsp 27 levels by an

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48 additional 25%. Hsp 27 was similar between the Con and ConH and the Im and ImH groups. Hsp 72 followed a very similar pattern when compared to hsp 27 in that there was a significant reduction in hsp 72 levels following immobilization (Figure 4-8). This reduction appears to be offset by reloading, as R7C did not differ from Con. Heating during reloading increased hsp 72 by an additional 30%. Hsp 72 content was nearly doubled in the ConH compared to the Con group and increased by 75% in the ImH group compared to the Im group. Hsp 32 behaved in a manner that differed greatly from the other hsps (Figure 4-9). Immobilization actually increased hsp 32 content by 32%, while reloading returned hsp 32 content to levels that were similar to Con. Reloading with heat increased hsp 32 levels by 37% when compared to Con and 25% when compared to R7C, but did not differ from Im. Surprisingly, heating did not increase hsp 32 content in the ConH group when compared to the Con group. Heating resulted in a 25% increase (p=0.06) in the ImH group when compared to the already elevated Im group. The content of all hsps in R7H was significantly higher than R7C. Con Im R7con R7H 0 1 2 #*#*Arbitrary OD Units Figure 4-7. Relative hsp 27 content following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H). Data is presented as means SEM. represents different from Con; represents different from R7H; # represents different from R7C.

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49 Con Im R7C R7H 0.0 0.5 1.0 1.5 ##*Arbitrary OD Units Figure 4-8. Relative hsp 72 content following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H). Data is presented as means SEM. represents different from Con; represents different from R7H; # represents different from R7C. Con Im R7C R7H 0 5 10 15 20 25 30 35 40 45 # #ng/ml Figure 4-9. Hsp 32 (HO-1) content following one week of immobilization (Im), immobilization followed by one week of reloading (R7C), or immobilization followed by one week of reloading in combination with a heat treatment (R7H). Data is presented as means SEM. represents different from Con; represents different from R7H; # represents different from R7C.

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CHAPTER 5 DISCUSSION This investigation sought to establish that providing a heat intervention during muscle reloading following one week of immobilization would augment muscle regrowth. To that end, four hypotheses were tested including: (1) Heat treatment will cause an increased muscle regrowth rate following eight days of reloading; (2) Heat treatment will reduce oxidative damage to both lipids and proteins during reloading; (3) Heat treatment will result in a reduction in antioxidant enzyme activity during reloading; (4) Heating will cause an increased activation of IGF-1 pathway intermediates during reloading. This is the first investigation demonstrating that heating will reduce oxidative damage during reloading and accelerate muscle regrowth. It was also demonstrated that the antioxidant protection was not due to antioxidant enzymes, which further implicate heat shock proteins as responsible for the observed antioxidant effect. Lastly, this is the first investigation to show that heating does affect IGF-1 pathway signaling during reloading, which may help to explain why muscle regrowth occurred at a higher rate in the R7H group when compared to the R7C group. Muscle Mass In support of the first hypothesis, heat treatment did augment muscle regrowth following one week of immobilization. Soleus muscles in the R7H group were significantly larger, both absolutely and when normalized for body weight, than soleus muscles in the R7C group. Potential support for this finding can be found from a recent study by Goto et al (76). In this investigation, there does not appear to be a difference in 50

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51 soleus wet weight between a reloaded and a reloaded heated group. However, when normalized for body weight, it does appear that the heated reloaded group is approximately 15% larger following seven days of reloading despite the fact that there is also no difference in body weight. Resolution for this apparent discrepancy could be that in the Goto study (76), only one heating bout was given during the 10 day reloading phase. Furthermore, they report only that hsp 72 is elevated while ignoring other heat shock proteins that may also be important in muscle regrowth augmentation including hsp 27 and 32. It is quite possible that both of these proteins may have fallen to control levels during the ten days of recovery following the lone heating bout. As the protective effect was minimal, if at all present, it seems important to consider that elevations of other or additional proteins beside hsp 72 may be required in order to maximize the potential of heat therapy in skeletal muscle regrowth augmentation. Several other studies have investigated the effect of heat on hypertrophy or cell culture growth. Seven days following a single heat stress (41C for 60 min) soleus muscles were larger when compared to a control group (106). Another study, using a similar design (41-42C for 30 min) showed an increase in soleus wet mass normalized for body weight as well as dry muscle weight in a heated group when compared to a control group (230). Lastly, it has been shown that heat increased protein content in rat skeletal muscle cells (L6) when compared to a control (77). Only one investigation has attempted to augment muscle regrowth following disuse using an antioxidant (110). In that study, the degree of atrophy after one week of reloading in the reloaded control group was approximately 45%, while the vitamin E supplemented reloaded group was approximately 30%. Stated in these terms, the degree

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52 of atrophy in this investigation in R7C was approximately 70%, while the degree of atrophy in R7H group was approximately 40%. This indicates that heating performed at least as well in assisting muscle regrowth as exogenous vitamin E. In another investigation, animals were supplemented with torbafylline in an attempt to decrease muscle atrophy during 5 wks of immobilization and enhance muscle regrowth during four weeks of reloading (1). Torbafylline is a xanthine derivative with a yet to be defined mechanism of action. In various conditions, it has been reported to function as an antioxidant, regulator of inflamation, and inhibitor of the ubiquitin proteasome pathway (1, 46). It failed to protect muscle mass during immobilization and was not effective as an agent to enhance hypertrophy during regrowth. It may be the reason it seemed so unsuccessful was that the time of immobilization was quite long at five weeks and the time of reloading was also quite long at two and four weeks. Any beneficial effect could have well been overcome by the severe state of atrophy and missed during reloading due to the prolonged time period of regrowth. Torbafylline was able to return mitochondrial density to control values and enhanced fatigue resistance during the reloading period. Oxidative Stress and Antioxidant Enzymes In the present study, heating significantly reduced oxidative stress caused by reloading in both lipids and proteins, in support of hypothesis 2. Furthermore, this reduction returned oxidative stress markers to control levels indicating that heating is a powerful antioxidant. In another study, it was shown that antioxidant supplementation both enhanced muscle regrowth and reduced oxidant damage following immobilization (110).

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53 In a previous investigation, using a similar heating paradigm, we have shown that heating can reduce oxidative damage during immobilization (as well as attenuate muscle atrophy) (201). Further, other investigations have demonstrated that heating can be a potent antioxidant intervention in a host of free radical generating conditions. In one investigation evaluating heart function and infarct size following an IR injury, heated hearts performed significantly better and had smaller infarcts sizes when compared to hearts that were not heated (100). In several studies meant to simulate peripheral vascular disease, IR injury of the limb was reduced with heating (72, 165). In this investigation, lipid oxidation was measured by assessing the ferrous oxidation of xylenol orange as well as the total HNE products. HNE was significantly increased in the Im group as well as the R7C group when compared to both Con and R7H, while the results of the FOX assay remained unchanged, regardless of treatment group. While they both are used as indices of lipid oxidation, what they measure is subtly different. The FOX assay measures total lipid hydroperoxides, likely from polyunsaturated fatty acid oxidation; however, HNE is a measure of lipid peroxidation end products that have reacted with proteins and left a characteristic fingerprint (183). Furthermore, the FOX assay, while a vast improvement over the TBARS assay, is still quite a bit more variable than the more stable HNE measure. The fact that one is different while the other remains unchanged should not suggest that the increase in lipid oxidation is tenuous, but that total hydroperoxides were not different; however breakdown products were elevated in the Im and R7C groups when compared to the Con and R7H groups.

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54 Analysis of the protein oxidation data is similar to that of the lipid oxidation data in that one measure was able to detect a difference, while another was not. The measure of protein carbonyls is a very precise assay that allows the quantification of proteins that have been attacked by toxic aldehydes or ketones. Nitrotyrosine, on the other hand, is a measure of nitrosylated tyrosine residues as a result of NO or ONOO attack. These data may indicate that NOS dysfunction may play a more critical role in free radical generation in this model than previously thought as both NO and ONOO ultimately originate from NOS. That one measure was different while the other remained unchanged should not suggest that the increase in protein oxidation is tenuous, but that protein carbonyls were not increased; however, nitrosylated tyrosine residues were elevated in the Im and R7C groups when compared to the Con and R7H groups. Because heating reduced oxidant damage, it becomes important to assess antioxidant enzyme status. In this regard, CuZnSOD and MnSOD, catalase, glutathione peroxidase, and glutathione reductase activities were measured. As there is no information regarding antioxidant enzyme activity during reloading in the literature, our hypothesis regarding reduced antioxidant enzyme activities in the R7H group when compared with the R7C group is based on the notion that there is a strong record of oxidant stress associated with reloading and pliometrically damaged skeletal muscle. In many instances, increases in oxidant stress are countered by increases in antioxidant enzyme activities. MnSOD, GPX, and GRX activities did not change in response to immobilization or reloading in conjunction with what has been found previously (111, 115, 201). The possible exception to this is GRX activity, which, in separate investigations, did not

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55 change in response to immobilization and increased in response to immobilization (111, 115, 201). Possible resolution to the incongruent findings, is that different rat strains used in these investigations produced dissimilar results. Furthermore, MnSOD activity was not increased in response to immobilization or reloading, which could indicate that mitochondrial production of O2is not elevated in these conditions (12, 84). From our data, we were able to show that CuZnSOD activity was increased in response to immobilization and remained elevated during reloading; however, reloading in combination with heat reduced CuZnSOD activity compared to control. This is in good agreement to what has been found previously during immobilization or unloading (111, 115, 124, 201). Because CuZnSOD activity is induced in response to O2-, it may indicate elevations in O2content in the cytosol of Im and R7C animals (12, 84). Moreover, it may also indicate that there is a reduction in O2in R7H animals when compared to the Im and R7C animals. In addition, immobilization and reloading resulted in an elevated catalase activity when compared to control, while reloading in combination with heat was similar to control. This is in good agreement with what has been found previously during immobilization and unloading (111, 115, 201). Both of these instances demonstrate support for hypothesis three in that heating during reloading resulted in a reduction in antioxidant enzyme activity. As antioxidant enzyme activities were reduced in the R7H group when compared to the R7C group, and oxidant damage, and presumably O2content, were reduced in the R7H group when compared to the R7C group, alternative antioxidant moieties must be considered (12, 84). Heat Shock Proteins Changes found in this investigation involving hsp expression following either Im or reloading appear to be typical of what has been found previously. Hsp 72 is by far the

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56 most studied of the hsps, and as such, provides the most data for comparison. Following unloading or immobilization, most studies show a reduction in either hsp 72 mRNA content or protein expression (19, 164, 215) while others have detected no change (172, 201). A previous suggestion was that the gender of the animal was the primary determinant in either a reduction or no change in hsp 72 mRNA content/ protein expression as only one study involving male rats was able to detect a reduction in hsp 72 expression (173). As this work represents the second investigation using males to detect a reduction in hsp 72 expression, this notion should be reconsidered. Reloading resulted in a sharp increase in hsp 72 expression following the reduction seen during immobilization in this data set. An increase in hsp 72 expression during reloading has been found previously (76, 173). Furthermore, reloading resulted in a four-fold increase in hsp 72 mRNA in only four hours of ambulatory recovery (19). In the present study, heating in combination with reloading further increased hsp 72 levels to that above reloading alone. It appears as though a similar result was found in another study (76). Hsp 27 has been largely overlooked in the unloading/reloading literature. In this investigation, immobilization resulted in a reduction in hsp 27 expression, while reloading resulted in a dramatic increase, to levels above Con. Heating combined with reloading increased hsp 27 expression further than reloading alone. As this protein has been largely ignored with regard to unloading/reloading, it is difficult to interpret this finding. In regard to unloading/reloading work, hsp 32, like hsp 27, has also been overlooked. In this investigation, immobilization resulted in a large increase in hsp 32

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57 expression. Previous work supports the notion of an increase in hsp 32 expression following disuse (93). As hsp 32 is increased in response to free heme, it may indicate that myoglobin is being degraded due to free radical attack (142, 143). Furthermore, there is evidence that during periods of oxidant stress, additional sources of heme may be available. These include cytochrome C, newly synthesized heme, and hemoglobin (142, 145). Newly synthesized heme is particularly vulnerable to catabolism by hsp 32 because during periods of oxidant stress the reduction in protein synthesis may mean that the macromolecule it was going to join will no longer be synthesized leaving it available for hsp 32 activity (142). Lastly, there is also a likelihood that with lipid oxidation, as in this model, hemoglobin can leak into the cell and ultimately contribute to the free heme pool (144, 146). With reloading, hsp 32 content was returned to baseline, while hsp 32 content in the R7H group reached that of Im. Given the similarities between immobilization and reloading in terms of oxidant stress, it is surprising that in one condition hsp 32 is induced, while in the other it is at control levels. It may be that in the environment of an atrophying muscle, the availability of newly synthesized heme is quite large (142, 145). By comparison, the environment of a reloading muscle is characterized by increases in protein synthesis that may reduce the free heme pool. As the antioxidant enzymes were not elevated in the R7H group and the oxidant damage was lower in this group, alternative antioxidant substances must be considered. Implicit throughout this project is the notion that heat shock proteins convey the antioxidant protection found through heating. Importantly, hsps were increased in each case in the R7H group when compared to the R7C group indicating that they may be responsible for the observed antioxidant effect. The antioxidant capabilities of many of

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58 these proteins are quite extensive. For example, in cells over-expressing either hsp 27 or 72, cell survivability and damage was significantly less when compared to control cell types (109, 238). Indeed, animals over-expressing hsp 72 recovered force significantly faster following a lengthening contraction protocol than did wild-type animals (147). Bilirubin, a product of hsp 32 activity, is a potent antioxidant and, like hsp 27 and 72, will prevent cell death and damage when an oxidant stress is encountered (68). Furthermore, gene array studies have shown that the only discernable trend following heating is an increase in heat shock proteins. Comparatively, these studies demonstrate inconsistent changes in other antioxidant substances including MnSOD, CuZnSOD, catalase, and various glutathione handling enzymes (192, 210, 211, 243). For example, in one investigation glutathione-S-transferase and MnSOD are reduced, while in another, both are increased following heating (192, 243). Nevertheless, the possibility remains that proteins other than hsp 27, 32, or 72 are responsible for the observed antioxidant effect. IGF-1 Pathway Muscle growth in the soleus following disuse atrophy appears to occur in two phases. The first, lasting approximately seven days, is dependant upon the IGF-1 pathway, while growth thereafter is reported to be dependant upon calcineurin and satellite cells (20, 158). As the regrowth period in this investigation was limited to one week, IGF-1 pathway activation was assessed. In this pathway, IGF-1 leads to the activation of three parallel pathways mediated by Akt. Down stream of Akt, Gsk phosphorylation decreases its inhibitory role on protein synthesis and mTOR activation results in the phosphorylation of p70 s6k which increases its protein synthesis role. In

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59 order to evaluate IGF-1 pathway activation, total Akt and phospho-Akt, total p70s6k and phospho-p70s6k, and total Gsk and phospho-Gsk were measured. Immobilization resulted in a reduction in total Akt followed by an increase in total Akt to that of nearly two fold Con in the R7C group. In another investigation, 10 days of unweighting resulted in a trend toward a reduction Akt content. During reloading, total Akt content was increased, like in the present investigation (43). Heating appeared to blunt the increase during reloading, as total Akt was increased to Con levels, but not beyond. Phospho-Akt content followed a similar pattern to that of total in that there was a reduction during the immobilization phase that was met by a strong induction in the R7C and R7H groups that did not differ. That phospho-Akt was down with immobilization and increased in response to reloading are in good agreement with other studies of similar design (43, 219). As total Akt was increased in the R7C group more than in the R7H group and the phospho-Akt content did not differ, a greater proportion of Akt must have been phosphorylated in the R7H group, thus, an increase in IGF-1 pathway activation could be indicated. Previous work has shown an increased Akt activation with heat shock, likely mediated through hsp 32 and 72, but not hsp 27 (64, 118, 202). Furthermore, hsps 32 and 72 have maintained Akt activity in the face of oxidant stress (64). P70 s6k content was reduced with immobilization and increased similarly in the R7C and R7H groups. P70 s6k phosphorylation followed a similar pattern as total content; however, immobilization did not cause a change in p70 s6k phosphorylation while it was increased similarly in the R7C and the R7H groups. In a similar investigation, unloading did not reduce phospho-p70 s6k content, and reloading caused a significant increase in

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60 phospho-p70 s6k content indicating that the present study and that mentioned are in good agreement (219). It does appear though, that the increase in p70 s6k content is approximately 20% larger in the R7C group when compared to the R7H group. Further, the phosphorylation of p70 s6k is approximately 20% greater in the R7H group when compared to the R7C group. While these differences are not statistically significant, these findings potentially support the notion that there is increased IGF-1 pathway activation in the R7H group when compared to the R7C group. This idea is further supported by the analysis of the Gsk and phospho-Gsk data. Immobilization did not cause a reduction in total Gsk content; however, reloading caused a nearly two fold increase in the R7C and R7H groups. These findings are in good agreement with previous work from Booths lab using a study of similar design (43). Like total Gsk, phospho-Gsk did not change in response to immobilization. Similar results have been found previously (43). Phospho-Gsk was also not increased during reloading; however, reloading in combination with heat resulted in a two-fold increase in phospho-Gsk content. In a previous investigation, Childs et al. (43) showed that reloading resulted in an increase in Gsk phosphorylation. Resolution to this apparent discrepancy remains elusive. As the present investigation represents only the second such study, and such divergent results were found, additional testing of this variable should be considered. In another investigation, heat shock increased Gsk phosphorylation during oxidant stress compared to non-heated cells (202). These findings support the premise that heating, and perhaps hsps specifically, reversed IGF-1 pathway dysfunction seen with reloading. This is particularly true in light of evidence that IGF-1 pathway activation has shown to be preserved by heat stress or

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61 hsp induction in the face of oxidant stress, as in the case with reloading (64, 118, 202). Furthermore, because muscle regrowth is IGF-1 dependant during this phase of hypertrophy, these data may contribute toward an understanding of why the muscle mass in the R7H group was significantly larger than the muscles in the R7C group. Integration In this investigation, the global hypothesis tested was that heating will enhance muscle growth following immobilization. Ultimately, this is based on results from Kondo et al. (110) who were able to use vitamin E to increase the rate of regrowth in a similar study design. That study also showed that there is an increased amount of oxidant damage with reloading that is attenuated with vitamin E supplementation. These authors conclude that a component of muscle regrowth is inhibited by oxidant stress and that exogenous antioxidants will provide some help. We, and others, have shown that various hsps, and heating in general, can act as an antioxidant, thus framing the initial belief in this project (72, 164, 201, 238, 239). The notion that heating, or heat mediated induction of hsps, could enhance muscle regrowth was further strengthened by the work of McArdle et al. (147) who were able to show that rats over-expressing hsp 72 recovered at a faster rate than wild type rats following an injury protocol that is similar to what is experienced during reloading. That muscle weights were larger in heated reloaded rats compared to reloaded rats supports the hypothesis that heating augments muscle regrowth. Furthermore, heating acted as a potent antioxidant in this model, returning lipid oxidation and protein oxidation to control levels. This further supports the notion that heating acted as an antioxidant and as such increased the rate of hypertrophy in a similar fashion to the work of Kondo et al. (110). Assessment of the antioxidant enzyme activities revealed that they are not

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62 responsible for the antioxidant protection; hence, alternative sources must be sought. We have previously found similar results in an immobilization study that used heat as a countermeasure to oxidant stress and muscle atrophy (201). It is speculated that hsps 27, 32, and 72 are conferring the antioxidant effect seen in the heated reloaded group. The design of this study prohibits stating conclusively that these proteins are responsible. Nevertheless, these proteins are increased in expression in the R7H group compared to the R7C group and can act as antioxidants. Furthermore, analysis of gene array studies reveals inconsistent changes in many other antioxidant substances (192, 210, 211, 243). The study design does allow for the conclusive statement that heating is an antioxidant intervention, independent of antioxidant enzymes, and that reduction of oxidant damage results in enhanced skeletal muscle regrowth. The last portion of this study dealt with the possibility that heating may enhance regrowth by affecting the activation of the IGF-1 pathway, possibly due to hsp interaction with IGF-1 pathway proteins. Previous work has shown that hsp 32 and 72 will increase Akt activity as well as increase Gsk phosphorylation during exposure to oxidant stress such as is the case with reloading (64, 118, 202). Akt comes early in the IGF-1 pathway and the results indicate that there is an increased activation of this protein in the R7H group compared to R7C as suggested by previous work (64, 118, 202). Down stream of Akt are both p70 s6k and Gsk in divergent pathways. Gsk is directly down stream of Akt and the results clearly show an increased phosphorylation of this protein, leading to an inhibition of its anti-synthesis function in the R7H group compared to the R7C group as has been found previously (202). P70 s6k also lies down stream of Akt (Akt/mTOR/p70 s6k ) and shows the familiar trend of an increased activation in the R7H group compared to

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63 the R7C group. It could be that heating acts to reduce oxidant stress, which in turn helps to maintain IGF-1 pathway fidelity, or that the reduction in oxidative stress seen with heating and the increased IGF-1 activation are independent events contributing to the same ultimate outcome increased muscle mass in a heated reloaded group when compared to a group that is reloaded without intervention. In summary, we have shown that heating will increase the rate of hypertrophy following one week of immobilization. Further, we are the first to demonstrate that heating will reduce oxidant damage in this model, in an antioxidant enzyme independent fashion. Lastly, we show that heating during reloading will help to maintain IGF-1 pathway activity. We suggest that hsps 27, 32, and 72 are likely responsible for the reduction in oxidant damage as they are induced in the heated reloaded group while inconsistent changes have been found for various other antioxidants following a heat stress. Additionally, we believe it possible that hsps 32 and 72 may be interacting with IGF-1 pathway intermediates to promote increased pathway stability. The culmination of reduced oxidant damage and increased IGF-1 activity likely caused the increased muscle mass found in the reloaded heated group when compared to reloading alone.

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BIOGRAPHICAL SKETCH Joshua Taylor Selsby grew up in Dayton and rural central Ohio before graduating from Mt. Gilead High School in 1995. He then attended the College of Wooster for his undergraduate education where he was a member of the mens swimming team for four years. During his time there he completed a thesis that combined his love of science with love of swimming entitled Swim Performance following Creatine Supplementation in Division III Athletes and was advised by Drs. Michael Kern and Keith Beckett. He graduated in May 1999 with a BA. Following graduation, he attended The Ohio State University in the Exercise Physiology Department under the mentorship of Dr. Steven Devor. His thesis was titled A Novel Mg-creatine Chelate and a low Dose Creatine Supplementation Regimen Improve Work. After graduating in June 2001 with an MA he attended the University of Florida under the supervision of Dr. Stephen Dodd in the Muscle Physiology Lab. While at UF he had the opportunity to be involved in several different projects before developing a keen interest in heat shock proteins. His thesis was titled Does Heat Treatment Facilitate Muscle Regrowth following Hind Limb Immobilization. Upon defense of his dissertation, Joshua joined the lab of Dr. Lee Sweeney at the University of Pennsylvania as a post-doc so that he could continue to develop as a scientist and learn new techniques to better explore skeletal muscle adaptation. 84


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DOES HEAT TREATMENT FACILITATE MUSCLE REGROWTH FOLLOWING
HIND LIMB IMMOBILIZATION?















By

JOSHUA SELSBY


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Joshua Selsby

































This document is dedicated to my favorite Anatomy student.















ACKNOWLEDGMENTS

First and foremost, I would like to thank my wife for her patience and

understanding. I also thank my parents who have provided much needed encouragement

and support over the years. The Muscle Physiology Lab group, especially Andrew Judge,

Sara Rother, Om Prakash, and Shige Tsuda, certainly receive my thanks and gratitude. I

thank my committee, Scott Powers, David Criswell, and Glenn Walter, for their time and

thought into this project. Lastly, I would like to thank my advisor, Stephen Dodd, who

has helped guide me to the completion of this project.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ......... ................................................................................... iv

LIST OF TABLES ............. .......... ..................... vii

LIST OF FIGURES ......................... .. .... ...... ............. viii

A B ST R A C T ................. .......................................................................................... x

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

B a c k g ro u n d ................................................................. .................................... 1
Q u estion s .......................................................................... . 4
H ypotheses ................................................ 4

2 REVIEW OF LITERATURE ......................................................... .............. 5

Significance .............................................................. .. .. ........ 5
Free R adicals and Oxidant Stress ........................................ ........................... 6
F ree R radicals ............................................... ......................... 6
Sources of Free R adicals ...................... .................................... .............. 7
Sources of Free Radicals During Immobilization ............. ..................................8
Regulation of Free Radicals ........... ..... ......... ...................9
Skeletal Muscle and Unloading ......... ............... ................... 10
M models ............. ........ ... ............... .......... 10
Changes with Disuse Atrophy .................................. ....................12
Oxidative Stress, Antioxidants and Unloading .................................................14
Skeletal M uscle and Reloading ............................................................................ 15
Adaptations to Reloading .......... .... ...................... ..... ........... .... 15
P athw ay s of R egrow th .............................................................. ..................... 16
Reloading: Injury and Damage....... .................. .............. ...................17
Reloading and the Immune Response ...................................... ............... 18
O xidant D am age ................................................................ .. ....... .. ...... 19
H eat Shock Proteins ............... ............ ........ ........ ...... .......... .. ................. 21
O v erv iew ....................................................... 2 1





v









H eat Shock Proteins 27, 32, and 72..................... ............ ............... ... 22
Heat Shock Proteins, Oxidant Stress, and Potential as a Reloading
In terv en tio n .................................................................. ............... 2 4

3 M E T H O D S ........................................................................................................... 2 8

D e sig n ............................................................................................................2 8
G general Procedures ......................................................... .. .. ... 29
Im m obilization and R loading ........................................ ........................ 29
Heat Treatment ....................... ..................... 30
Muscle Removal and Sample Preparation............................... ......... ........31
B ioch em ical P rocedu res .............................................................................................32
W e ste rn B lo t .................................................................................................. 3 2
G lutathione Peroxidase............................................... ............. ............... 33
G lutathione R eductase...................... .. .. .......... .. ..................... ............... 34
Catalase ................ .................................34
Superoxide D ism utase ............................................................ ...............35
Ferrous Oxidation Xylenol O range .............................................................. 35
E L I S A S .......................................................................................................... 3 6
Statistical A analyses .................................................. ...... ............ ... 36

4 R E S U L T S .............................................................................3 7

W hole B ody M easures............................................. ................... ............... 37
W hole M uscle M easures........................................... ........................................ 8
Oxidative Damage ................................. ... ........ ................. 40
A nti-O xidant E nzym es ....................................................................... ..................42
IG F -1 P athw ay A ctivation ............................................................... .....................43
H eat Shock Proteins ........ ..... .................... ............. .. .........47

5 D ISC U S SIO N ......... ......... ............................................. ........................... 50

M u scle M ass ................... ........................................ ................. 50
Oxidative Stress and Antioxidant Enzym es ............................................. .............52
Heat Shock Proteins....... ....... ......... .................. .......... ................. 55
IGF-1 Pathway ............... ......... ................ 58
Integration ........... .... .............. ................................... ...........................6 1

L IST O F R E FE R E N C E S ........... ........................................................ .......................... 64

B IO G R A PH IC A L SK E TCH ..................................................................... ..................84
















LIST OF TABLES


Table p

4-1 Body weight changes at various time periods during this investigation. Values
are shown in grams and are presented as means + SEM ........................................38

4-2 Relative muscle mass, water content, and wet weight to dry weight ratio in the
Con, Im R7C and R7H groups. ........................................ ......................... 40

4-3 Activities of various antioxidant enzymes in the Con, Im, R7C and R7H groups...43















LIST OF FIGURES


Figure p

2-1 Schematic of antioxidant scavenging system ................................... ... ..................10

3-1 Schem atic of study design ......... .................. .................................. ............... 29

3-2 Schematic of protocol for one hypothetical week......... .................................... 29

4-1 Wet muscle mass in control animals (Con), following one week of
immobilization (Im), immobilization followed by one week of reloading (R7C),
or immobilization followed by one week of reloading in combination with a heat
treatm ent (R 7H ) ................................................. ................. 39

4-2 4-hydroxy-non-enol as determined by immuno-blotting following one week of
immobilization (Im), immobilization followed by one week of reloading (R7C),
or immobilization followed by one week of reloading in combination with a heat
treatm ent (R 7H ) ................................................. .................. 4 1

4-3 Nitrotyrosine as determined by dot blot immuno-blotting (top) and conventional
western blotting (bottom) following one week of immobilization (Im),
immobilization followed by one week of reloading (R7C), or immobilization
followed by one week of reloading in combination with a heat treatment (R7H)...41

4-4 Akt content (top) and phosphor-Akt content (bottom) as determined by immuno-
blotting following one week of immobilization (Im), immobilization followed
by one week of reloading (R7C), or immobilization followed by one week of
reloading in combination with a heat treatment (R7H).......... ......... ...............44

4-5 Total Gsk (top) and phosphorylated Gsk (bottom) content as determined by
immuno-blotting following one week of immobilization (Im), immobilization
followed by one week of reloading (R7C), or immobilization followed by one
week of reloading in combination with a heat treatment (R7H)............. ..............45

4-6 Total p70s6k (top) and phosphorylated p70s6k (bottom) content as determined
by immuno-blotting following one week of immobilization (Im), immobilization
followed by one week of reloading (R7C), or immobilization followed by one
week of reloading in combination with a heat treatment (R7H)............. ..............47









4-7 Relative hsp 27 content following one week of immobilization (Im),
immobilization followed by one week of reloading (R7C), or immobilization
followed by one week of reloading in combination with a heat treatment (R7H)...48

4-8 Relative hsp 72 content following one week of immobilization (Im),
immobilization followed by one week of reloading (R7C), or immobilization
followed by one week of reloading in combination with a heat treatment (R7H)...49

4-9 Hsp 32 (HO-1) content following one week of immobilization (Im),
immobilization followed by one week of reloading (R7C), or immobilization
followed by one week of reloading in combination with a heat treatment (R7H)...49















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

DOES HEAT TREATMENT FACILITATE MUSCLE REGROWTH FOLLOWING
HIND LIMB IMMOBILIZATION?

By

Joshua Taylor Selsby

December 2005

Chair: Stephen Dodd
Major Department: Applied Physiology and Kinesiology

In order for skeletal muscle to return to its original state following immobilization,

reloading of the muscle is necessary. Reloading is characterized by a period of increased

oxidant damage to skeletal muscle macromolecules. Previous work has shown that

antioxidant supplementation during reloading will decrease oxidant stress and augment

the rate of muscle hypertrophy. We have previously shown that heating can provide a

potent antioxidant effect. It was hypothesized that heating in conjunction with reloading

would result in a faster rate of hypertrophy than reloading alone.

In order to test this hypothesis, male Sprague-Dawley rats were divided into control

(Con), immobilized (Im), reloaded (R7C), and reloaded heated (R7H) groups

(n=10/group). Animal hind limbs in the Im, R7C and R7H group were plantarflexed and

immobilized bilaterally for one week. At the conclusion of one week, Im animals were

killed and reloaded animals were returned to their cages for one week. Beginning 24 hrs.









prior to reloading and continuing on alternating days, animals in the R7H group were

heated such that the core temperature was sustained at 41.50C for 30 min.

Immobilization resulted in a reduction in muscle mass that was not corrected by

reloading alone; however it was significantly increased in the R7H group. In addition,

oxidant stress to lipids and proteins was increased in the Im and R7C groups; however,

reloading in combination with heat returned both markers to Con levels. Assessment of

the antioxidant enzymes revealed that they are not responsible for the observed

antioxidant effect of heating. Heat shock proteins 25, 32, and 72 were increased in the

R7H group when compared to the R7C group indicating that they may be responsible for

the observed antioxidant effect. Lastly, the R7C group may suffer from an IGF-1

pathway dysfunction that is corrected in the R7H group.

The summation of this data is that heating augments muscle regrowth following

immobilization. Further, it reduced oxidant damage via an antioxidant enzyme

independent mechanism, and likely through a heat shock protein dependant mechanism.

Lastly, heating corrected the IGF-1 dysfunction seen during reloading.














CHAPTER 1
INTRODUCTION

Background

As life expectancy continues to expand, the incidence of infirmity and the

frequency of immobilization will increase. In addition, our increasing emphasis on

prolonged space travel will require an extended exposure to microgravity. Regardless of

method, severe atrophy of the locomotor muscles will follow. Much research has been

dedicated to the study of unloading and development of countermeasures to this type of

muscle loss. What has been neglected, however, is the need to reload these muscles

following unloading. The present study is designed to investigate a potential

countermeasure to damage that occurs during the reloading process.

Disuse atrophy induced via casting has been repeatedly demonstrated to reduce

skeletal muscle mass, cross sectional area, and force (8-10, 22, 69, 100, 108, 112, 197,

219). Furthermore, upon reloading, the atrophied muscle undergoes damage much like

that seen with a pliometric (eccentric) contraction (69, 94, 100, 227). Accompanying this

damage is an increase in the oxidative stress encountered by the reloaded muscle (108,

236). It has also been shown that supplementation with an antioxidant can reduce the

oxidative stress and lessen damage observed with reloading and hasten muscle regrowth

(108).

The concept of reloading injury as being caused by a pliometric contraction is not a

new idea. In fact, in normal cage sedentary activity the ambulation cycle contains a

lengthening contraction component (131). When the hind limb is immobilized in the









shortened position, severe atrophy of the soleus occurs, both in terms of mass and length

(27, 49, 186). As the soleus is the primary antigravity muscle and ambulatory muscle of

the hind limb, it is not surprising that it undergoes contraction-induced injury upon

reloading especially when it is considered that a shortened muscle will be required to

work through the range of motion of its previous length. Furthermore, it is possible that

reloading causes the muscle's load-bearing capacity to be exceeded (100). Histologically

this damage can be observed as irregular widening of sarcomeres, A band disruptions,

and Z-line streaming (65, 184, 185, 187). Further, injured muscle may also have

centralized nuclei and membrane disruptions (42, 164, 165, 223).

The cause of this damage initially was thought to be mechanical in nature. Clearly

a mechanical component exists as myosin and titin disruptions have been observed (221,

237). It is now apparent that a secondary oxidative stress also occurs during reloading

and may exacerbate the damaged condition within the reloaded fiber (108). It seems

likely that this secondary oxidative stress is occurring due to several factors involving a

loss of Ca2+ homeostasis and containment within the cell during reloading (21, 94). The

elevated intracellular Ca2+ can in turn activate Ca2+ sensitive proteins, likely calpains, that

convert xanthine dehydrogenase to the free radical producing xanthine oxidase resulting

in the production of super oxide (113, 146). A second source of free radical production

may be of mitochondrial origin. As the Ca2+ levels rise, more is transported into the

mitochondria via the uniporter, which has the effect of reducing membrane potential and

electron transport (82, 85). It also causes an increase in electron leakage and increases

superoxide production (39, 57, 85, 86). Finally, increased Ca2+ concentrations will

increase NO synthesis and the potential for ONOO* production (136). The production of









free radicals is made worse by the fact that the fiber already is in a state of compromised

oxidant resistance (113, 122).

From previous work, it can be concluded that countering this elevated oxidative

stress is beneficial to muscle regrowth as antioxidant supplementation resulted in larger

muscles following reloading (108). Hence, a potential mechanism to enhance muscle

regrowth following disuse atrophy may be the application of a heat stress causing the

subsequent over expression of heat shock proteins (hsps) and a concomitant reduction in

oxidant stress. There are several features of hsps, hsp 27, 32, and 72 in particular, that

make them attractive as potential candidates for this role. Together, these proteins can

scavenge free radicals, assist in the folding of nascent polypeptides, refold damaged

proteins, and assist in the clearance of proteins damaged beyond repair (72, 106, 120,

133, 163, 200, 211). By removing free radicals from the fiber, they are not available to

cause damage. By folding nascent polypeptides the elongation phase of protein synthesis

is actually enhanced resulting in a more rapid rate of protein synthesis. Refolding

damaged proteins will spare them from degradation. Finally, clearing damaged proteins

will prevent them from accumulating within the cell and cause a potentially cytotoxic

accumulation.

The notion of using a heat intervention in order to confer protection during in vivo

perturbations is not new as it has repeatedly been shown to be cardioprotective (77, 98) as

well as protective to skeletal muscle during an ischemia/reperfusion injury (124).

Heating has also been shown to attenuate muscle atrophy both by hind limb unweighting

(161) and by hind limb immobilization (197). In each of these cases, protection was

attributed to the over-expression of hsps. More recently, McArdle et al. (144) were able






4


to show that following pliometric injury, transgenic mice over-expressing hsp 72

recovered faster and did not suffer the well-characterized secondary force reduction.

Additionally, transgenic mice had a higher degree of undamaged muscle when compared

to wild-type. Thus, the present investigation is designed to examine whether heat stress

can decrease oxidative damage found within the cell while simultaneously increasing

hypertrophy during the reloading period.

Questions

The following questions were investigated in this study.

1. Will heating reduce oxidant damage to both lipids and proteins during reloading
after immobilization?

2. Will heating cause changes in the activities of native antioxidant enzymes during
reloading after immobilization?

3. Will heating accelerate the rate of muscle regrowth during reloading after
immobilization?

4. Will heating cause a greater activation of signals of protein synthesis during
reloading after immobilization?

Hypotheses

The following hypotheses were made in this study.

1. Heat treatment will reduce oxidative damage to both lipids and proteins during
reloading.

2. Heat treatment will result in a reduction in antioxidant enzyme activity during
reloading.

3. Heat treatment will cause an increased muscle regrowth rate following eight days
of reloading.

4. Heating will cause an increased activation of protein synthesis components during
reloading.














CHAPTER 2
REVIEW OF LITERATURE

Significance

Muscle atrophy is a critical health concern in several disease states and has an

impact on the quality of life and independent living in the elderly (156, 218). Much of

this atrophy occurs due to interventions, including bed confinement and cast

immobilization implemented by physicians in care of their patients. Regardless of the

cause of atrophy, reloading of the muscle will be necessary in order to return to a pre-

atrophy activity level. Much research has been conducted investigating mechanisms that

cause atrophy. Further, a host of countermeasures have been found that appear to be

effective in reducing atrophy. Comparatively little research has been conducted

investigating the potential for interventions that enhance the regrowing process during

subsequent muscle reloading.

This review will focus on muscle reloading, building toward a hypothesis for a

novel intervention during the reloading process. In order to fully appreciate changes in

skeletal muscle occurring during reloading it is important to discuss several related topics

as well. Oxidant stress increases during disuse atrophy and reloading; hence an

understanding of oxidative stress is relevant. Secondly, it is also important to understand

the condition of the muscle following disuse atrophy, as significant remodeling has

occurred. Finally, as relatively little research has been conducted encompassing some

topics of reloading, lengthening contraction data will also be included and discussed.

This is relevant because during normal ambulation there is a lengthening contraction









experienced by the hind limb muscles (134). Under normal conditions, the muscle has

adapted to this repeated pliometric contraction and, as such, it is not damaging. Upon

reloading following disuse, these repeated pliometric contractions can be quite

devastating to the muscle. As the damage inflicted comes as a result of lengthening

contractions, it becomes important to cull this literature of material relevant to the

discussion at hand (134). Finally, this review will propose the use of heat stress and

resultant over-expression of hsps as a means to augment the regrowing process.

Free Radicals and Oxidant Stress

Free Radicals

A free radical is a molecule or atom, existing independently, with an unpaired

valence electron (33, 54, 186). More specifically, reactive oxygen species (ROS) are

oxygen containing free radicals, superoxide (02*-) and the hydroxyl radical (OH.).

Reactive nitrogen species (RNS) are nitrogen containing free radicals and include nitric

oxide (NO.) and peroxynitrate (ONOO*). Some substances, such as hydrogen peroxide

(H202), are said to be pro-oxidant, indicating that they lend themselves to the production

of free radicals. Finally, there are substances called antioxidants that can scavenge free

radicals and process them to either a less deleterious state or remove them altogether.

While cells do require a certain redox balance in order to function, a shift in that balance

toward an oxidant state indicates oxidant stress. This can occur due to an increase in free

radical production, a decrease in antioxidants, or a combination of the two.

When cells experience oxidant stress, they can be expected to show signs of

oxidant damage to macromolecules including lipids, proteins, and nucleic acids (137,

207, 208, 235). In the case of lipids, the membrane bilayers are often vulnerable, leading

to membrane lesions and loss of chemical and electrical gradients that can occur at both









the cellular and organelle level (14, 37, 55, 88, 92, 124, 199, 206). Lesions in the

sarcolemma can cause a loss of Na+, K+, and Ca2+ homeostatic balance leading to a host

of deleterious effects. In the case of mitochondria, lesions can lead to cytochrome C

triggered apoptosis and loss of myonuclear number, as well as other harmful events.

Protein oxidation and damage have received a great deal of attention in oxidative

biology. The protein is acted on by a free radical and becomes oxidized resulting in

either minor or major damage to the protein. In the case of minor damage, the protein

can be repaired or refolded (17, 120, 174, 200). Severe damage results when the repair

mechanisms are not sufficient to return the protein to a functional state. Should this

occur, proteins are ubiquitinated and subsequently degraded by the 26S proteasome

(101). Alternatively, recent evidence has suggested that degradation of oxidant-modified

proteins can also occur by the 20S proteasome in a ubiquitin independent manner (51,

179).

Oxidative attack of nucleic acids can occur at either the DNA level or the mRNA

level (137). In the case of the former, modified bases may lead to errors during

replication or transcription. Should the error be passed on during replication the result

may be a non-functional gene in the daughter cell. This can lead to unchecked cell

division and potentially cancerous growth. If free radicals attack mRNA the gene

product will likely not be translated.

Sources of Free Radicals

There are several sources within the cell that can create free radicals. During

oxidative phosphorylation, molecular oxygen is the final electron acceptor resulting in the

production of two water molecules. Approximately 5% of oxygen; however, will result

in the production of 02*- and free radical products rather than water (39). These free









radicals will then begin to damage lipids, proteins, and nucleic acids. Xanthine oxidase

acting on xanthine and hypoxanthine can also produce 02*-. Superoxide is dismutated by

superoxide dismutase (SOD) into the more stable H202. While not a free radical itself,

H202 is very pro-oxidant, especially when exposed to Fe2+, leading to a series of

reactions termed Fenton chemistry (30). In short, these reactions involve iron interacting

with several moieties to produce the free radical, OH*.

The free radical NO* is used in the body for many things including as a

neurotransmitter and a regulator of vascular tone (34, 44, 104, 231). It is produced in

numerous tissues by several differing isoforms of NO synthase (NOS) including the

neural isoform, the endothelial isoform, and the immune cell produced isoform. Despite

its pluripotent biological roles, due to its unpaired valence electron, NO* is also a free

radical. When it interacts with 02, the result is the cytotoxic ONOO*, the second of the

RNS (36, 182).

Sources of Free Radicals During Immobilization

During immobilization, there are several events that occur that lead to the

production of free radicals. The first is an increase in the cytosolic Ca2+ concentration,

indicating a loss of Ca2+ homeostasis and balance (96, 97, 206). Calcium activates

proteases that catalyze the change from xanthine dehydrogenase to the free radical

producing xanthine oxidase (149, 163, 214). Additionally, free iron also increases within

the cell, permitting the formation of OH* (30, 113, 114). Indeed, elevated OH* levels

have been measured in immobilized hind limbs (116).

Alternative pathways leading to the production of free radicals may exist. When

cytosolic Ca2+ levels are high, the mitochondrial uniporter will begin to transport more

Ca2+ than it can expel resulting in elevated mitochondrial Ca2+ concentrations (83, 98).









As a result, the membrane potential is decreased and rate of electron transfer in the

electron transport chain is reduced, potentially allowing a greater amount of electron

leakage at complexes I and III and subsequent O2- production (39, 58, 87). This is

compounded by the fact that enzymes responsible for the removal of 02*- are reduced

during disuse (115, 124). Finally, elevated Ca2+ within the cell or mitochondria can

increase the activity of NOS, leading to an increase in NO* production (139). As

mentioned earlier, NO* can interact with 02 and produce the highly cytotoxic ONOO*.

Further compounding matters is the fact that as more free radicals are produced, more

Ca2+ will be released from the SR, thus beginning a positive feedback loop where ROS

propagates Ca2+ release, while inhibiting Ca2+ removal, and high intracellular Ca2+ is

facilitating ROS production (2, 79, 80, 194, 223).

Regulation of Free Radicals

In order to combat free radicals and oxidative stress, the body has several potent

antioxidant enzymes and a host of other antioxidant molecules. As mentioned earlier,

SOD catalyzes the formation of H202 from 02*- by combining it with water. MnSOD is

responsible for the dismutation of mitochondria mediated O02- production, while

CuZnSOD is responsible for cytosolic 02*-.

Hydrogen peroxide has itself been demonstrated to be harmful to the cell due to its

instability; hence its removal is required. Glutathione peroxidase (GPX) decreases the

activation energy of the reaction between glutathione (GSH) and H202 resulting in the

formation of water and oxidized glutathione (GSSH). Glutathione reductase can then

reduce GSSH so that it can interact with H202 once again. Catalase is also responsible

for the breakdown of H202, although it needs no other molecule to perform this role.









There are other substances that are free radical scavengers that are not antioxidant

enzymes. The first of these was alluded to above GSH. Two GSH can interact and

scavenge one H202 resulting in the formation of a glutathione dimer. Additionally,

vitamins C and E have been demonstrated to be free radical scavengers. These

compounds, along with others, interact to form a cascade of free radical removal such

that some substances remove free radicals directly, while others, in effect, recharge

scavengers (Figure 2-1). A breakdown in part of the chain can greatly attenuate the

effectiveness of the antioxidants.

GRRSSNG!C NADP+


R Vitamin EC or GSH re e V n
Further down stream, NADPH reduces GSSG, while lipoic acid reduces

Vitamin C- DHT A
RSk l M e Vitaminnd Un


Vitamin C nTA
Figure 2-1. Schematic of antioxidant scavenging system. In this example, Vitamin E
scavenges the ROS, while either Vitamin C or GSH recharge Vitamin E.
Further down stream, NADPH reduces GSSG, while a lipoic acid reduces
oxidized vitamin C (provided by Karyn Hamilton, personal communication).

Skeletal Muscle and Unloading

Models

There are three common models used in research to induce atrophy. An obvious

similarity among these models is that they reduce the normal load the muscle experiences

and atrophy results (134). Two of these models are reduced use models, in which the

muscle is still capable of contracting; however, the force the animal can generate has

been greatly reduced. The third of these models involves transsection of the spinal cord,

and, as such, is a true disuse model. As the purpose of this review is a discussion









relevant to reloading, this review will focus only on models of reduced use, and

accordingly, spinal transsection will not be addressed. Disuse atrophy and reduced use

atrophy will be used interchangeably throughout. While this may technically be

inaccurate, disuse atrophy has been used colloquially to encompass both reduced use and

true disuse.

The two models of reduced use atrophy are hind limb suspension (HLS; it is also

commonly called hind limb unweighting, HLU) and hind limb immobilization (HLI).

During HLS, the animal is suspended by the tail such that the hind limbs are no longer in

contact with the cage bottom (7). The animal is generally given free reign over the cage

and can move by way of the forelimbs. In this model the hind limbs muscles are capable

of being recruited; however, they experience no load and produce no force.

During HLI, the animal's hind limbs are immobilized by cast fixation (22).

Generally, immobilization occurs in the plantar flexed position causing shortening of the

hind limb muscles and lengthening of the anterior limb muscles; however, some

investigators have made use of a neutral position or a dorsiflexed position. Muscle

groups immobilized in a shortened position experience significantly more atrophy than

muscles immobilized in a neutral or lengthened position, which will experience either no

loss of mass, or slight, but significant, hypertrophy (4, 26, 74, 99, 127, 171). During HLI,

the animal is capable of recruiting fibers in immobilized muscles; however, muscle EMG

activity is reduced by 85-90% ofuncasted controls (90). Further, the force generated

from a shortened muscle is not sufficient to prevent or even reduce atrophy when

compared to a muscle of a HLS animal.









Changes with Disuse Atrophy

During disuse atrophy the muscle undergoes significant remodeling, yielding a

smaller, less functional muscle. There are significant losses in mass and cross sectional

area (10, 124). There is also a disproportionate loss in force causing specific tension to

decrease, likely due to uncoupling of the excitation/contraction pathway (70). Mentioned

earlier was a loss of Ca2+ homeostatic containment and an elevation of intracellular Ca2

concentration (21, 96, 97, 206, 213). Further, the myosin heavy chain isoform should be

expected to shift toward the faster isoforms. This is especially pronounced in the

predominantly type I soleus. By shifting to faster isoforms, the muscle is better able to

maintain power when force generation is falling, as power is a function of both force and

contraction speed.

During disuse atrophy there is also an increase in protein degradation rates and

proteolytic components (52, 101, 125, 169, 198). While the precise mechanism of the

elevated protein degradation is currently unknown, some aspects have been defined. It

appears as though an early event in the pathway is the release of actin and myosin from

the sarcomere likely through calpain cleavage at the Z-lines (91, 229). Once the

contractile proteins are freed, they can then be ubiquitinated. The ubiquitination process

involves several enzymes, classified as El, E2, and E3, each performing a specific role

within the cell.

El is the ubiquitin activating enzyme and in an ATP dependant manner converts

ubiquitin from an inactive form to an active form (47). E2 is a ubiquitin conjugating

enzyme and carries activated ubiquitin to E3, ubiquitin ligase, that has already bound the

damaged protein (128, 131, 132). E3 attaches ubiquitin to the damaged protein. Two

isoforms of E3, atrogin (also called muscle atrophy F-box) and muscle ring finger-1









(MuRF-1), are believed to be involved in degradation specific to atrophy as they are

sharply elevated in several conditions causing atrophy (38, 75, 125, 152, 196, 198).

Once a poly-ubiquitin tail, consisting of four or greater ubiquitin molecules, has been

formed, the protein can be identified and degraded by the 26S proteasome (41, 227). The

protein is denatured on the surface of the proteasome in an ATP dependant manner and

subsequently broken down into polypeptides normally between 4 and 20 amino acids in

length (23). Within the core of the proteasome are five catalytic sites capable of breaking

peptide bonds including chymotrypsin-like activity (CT-L), trypsin-like activity (C-L),

peptidyl-glutamyl-peptide hydrolyzing (PGPH), branch-chain amino acid preferring

(BrAAP), and small neutral amino acid preferring (SNAAP) (175, 176). Each step, from

calpain activity to 26S activity, has been demonstrated to be elevated during disuse

atrophy.

In addition to increased degradation, there is a very rapid reduction in protein

synthesis occurring within hours of onset of disuse (121, 125, 225). There are three

phases to protein synthesis including initiation, elongation, and termination and are all

likely steps of regulation. It has been determined; however, that it is the elongation phase

that is inhibited and causes the reduction in protein synthesis during disuse. In an

elegantly designed study, Ku and Thomason (121) were able to determine differences

between the influences of initiation, elongation, and termination and conclude that

regulation of the elongation phase alone was responsible for compromised protein

synthesis. It seems regulation of several key proteins involved in elongation may be to

blame. Eukaryotic elongation factor 1A is responsible for the binding of tRNA to the

ribosome. The demethylation of eEF-1A, by unknown mechanisms, will slow the









elongation phase as tRNA will not be able to bind the ribosome (121). Eukarytotic

elongation factor 2 is responsible for translocation. Phosphorylation of eEF-2, via eEF-2

kinase, causes a slowing in the translocation step. Interestingly, oxidative stress will

cause an increase in the activity of eEF-2 kinase and a subsequent reduction in eEF-2

activity (180). Not only does eEF-2 kinase appear to be redox sensitive, but it also will

increase activity in a Ca2+/calmodulin dependant manner as well (25). In a hind limb

immobilized animal there is both an increased oxidative stress as well as an increased

intracellular Ca2+ concentration (96, 97, 116, 206). The summation of an increased

protein degradation rate and depressed protein synthesis rate yields a sharp reduction in

protein content within the cell (225). Protein content will continue to fall until a new

steady state level is met at approximately three weeks when protein degradation is no

longer elevated and protein synthesis has reached a new steady state.

Oxidative Stress, Antioxidants and Unloading

As demonstrated earlier, oxidative stress is clearly increased during periods of

unloading. It seems that elevated free Ca2+ leads to the production of 02*- via xanthine

oxidase or mitochondria, which can lead to the production of other free radicals (Figure

2-1). These free radicals can then damage lipids, protein, and DNA. In the case of

protein, the increased damage likely contributes to the increased protein degradation

observed with disuse atrophy; thus, if oxidative damage is reduced, damaged protein

content may be reduced, and, therefore, protein degradation rates may be reduced.

Further, if oxidant stress could be reduced, elongation rates may be maintained. This

leads to the hypothesis that supplementing with antioxidants during disuse atrophy may

protect the muscle from atrophy and may help to maintain protein content.









The antioxidant, vitamin E, has been utilized in several investigations with varying

degrees of success. Kondo et al. (112) supplemented animals with vitamin E and

reduced oxidative damage and increased muscle mass in HLI animals by approximately

20%, when compared to HLI alone. In addition, Appell et al. (10) found nearly 66%

larger cross sectional area in vitamin E supplemented animals following HLI than with

HLI alone. In another investigation; however, an antioxidant cocktail containing vitamin

E failed to preserve muscle mass during HLS (107). Despite the noted variability in

effectiveness, the rationale for using an antioxidant remains sound.

Skeletal Muscle and Reloading

Adaptations to Reloading

As stated earlier, skeletal muscle is a highly plastic tissue and will respond to a

change in workload by altering gene expression. In the case of reloading, skeletal muscle

is transitioning from a state of disuse to a state of increased workload. This is

accomplished by allowing the animal to re-ambulate, normally following hind limb

unweighting or hind limb immobilization. In a matter of days, there is a dramatic

increase in muscle mass, muscle cross sectional area, and force (140). Protein synthesis

increases rapidly upon reloading with actin and cytochrome C synthesis peaking at four

days (160). Protein degradation also increases dramatically during the initial stages of

reloading. After only 18 h of reloading calpain, ubiquitin, and the 20S proteasome

subunit mRNA are elevated (224). Additionally, both Ca2+ independent and non-

lysosomal protein degradation are elevated at 18 h (224). This continues up to one week

later as the remodeling phase involves the removal of damaged protein prior to full

recovery.









Depending upon the duration of disuse and age of the animal, complete recovery

may not occur with reloading. In adult animals, the muscle will atrophy and regrow as

expected. In the case of old animals, the muscle will atrophy; however, it will only

recover slightly (240, 241). Finally, in juvenile animals that have undergone an

unloading/reloading cycle, muscles generally do not reach the size of muscles in control

animals, even during adulthood (162).

Pathways of Regrowth

The mechanism of skeletal muscle regrowth appears to biphasic and fiber type

specific; however, it is still highly debated and researched. There does appear to be

consensus that during the first week of regrowth, the soleus regrows in a calcineurin-

independent and satellite cell independent manner and an insulin-like growth factor-1

(IGF-1) dependent manner (20, 158). During the second week of regrowth it appears the

soleus is dependent upon both calcineurin as well as satellite cells (158). The

mechanisms of regrowth of the plantaris are less clear as evidence has been collected

supporting both a calcineurin dependent and independent first week of growth, as well as

an IGF-1 dependent first week of growth (20, 158). It does appear that the second week

is calcineurin dependent and satellite cell independent (158).

IGF-1 activates phosphatidyl-inositol 3-kinase (PI3K) (56), which in turn activates

protein kinase B (PKB; Akt) through phosphoinositide-dependent kinase-1 and -2 (PDK

1 and 2) (31, 59, 105). PKB activates three parallel pathways culminating in elevated

protein synthesis. It has become clear in recent years that oxidant stress, heat shock, and

potentially hsps 32 and 72 (likely not hsp 27) specifically, will cause an increased

activation, i.e. increased phosphorylation, of IGF-1 pathway intermediates (56, 57, 64,

118, 202). Both oxidant stress and heat shock will increase PKB activation via PI3K,









although some debate remains regarding an alternative heat shock/PKB activation

pathway independent of PI3K and mitogen activated protein kinase activating protein-2

(MAPKAP-2) (117, 202). In addition, oxidant stress activation of PKB may be

dependant upon intracellular Ca2+ (50). These findings are of particular interest in a

reloading model as there is both an elevated oxidant stress as well as an elevated Ca2+ in

reloading skeletal muscle fibers (discussed below).

Reloading: Injury and Damage

During reloading, the formation of sarcomeric lesions on skeletal muscle fibers is

common and appears to increase in severity until approximately seven days of reloading,

supporting elevated protein degradation data presented earlier (102, 226). These lesions

are characterized by sarcomeric widening, myofibrilar loss, as well as myofibrilar

misalignment, and are localized rather than dispersed (71, 135, 141, 151). Lesion

formation is nearly instantaneous upon reloading and has been found to appear after only

one lengthening contraction (226). Further, it appears that the initial step in lesion

formation is damage to the sarcomere and perhaps more specifically, to myosin or titin

(226, 242). It also appears that the soleus undergoes more severe lesion formation than

the plantaris with the lesions occurring in 3-46% of fibers. Several reasons have been

proposed to explain the disparity and variability, including differing recruitment patterns,

cytoskeletal components of differing isoforms, and differences in contractile proteins and

properties.

To help clarify this issue, research has been conducted on reloaded muscle after

exposure to microgravity in space (232). This study was able to discern several different

fiber types within the same muscle including a slow phenotype as well as a hybrid

phenotype expressing both type I and II myosin isoforms. Ninety percent of type I fibers









had lesions on the sarcolemma, while only ten percent of hybrid fibers exhibited

sarcolemmal lesions. While the authors considered a variety of causes for the difference

in lesion formation between the two phenotypes, they cite difference in recruitment

pattern as the most likely source. They argue that even within the same muscle, a type I

fiber will be recruited more than a hybrid fiber. Accordingly, a type I fiber will be

subjected to more stress and have a greater potential for damage.

In addition to damage to the sarcolemma, the cytoskeleton also suffers damage

during reloading. The cytoskeleton is comprised of a host of proteins and is responsible

for transmitting force across the sarcolemma. This raises the possibility that, should the

network be ruptured, force production may suffer. Dystrophin undergoes severe damage

very soon after an eccentric contraction, primarily at the -COOH end (138). Meanwhile,

associated cytoskeletal components such as 0 spectrin and desmin are nearly unaffected

(138). It has been shown that during reloading or pliometric injury, there is a loss of Ca2

homeostasis within the cell and a subsequent increase in Ca2+ activated proteases,

including calpain (5, 6). The -COOH end of dystrophin is particularly sensitive to

calpain attack, which may help explain why dystrophin seems to be selectively damaged,

while other cytoskeletal components are not (48, 89, 212).

Reloading and the Immune Response

As is common with many types of muscle injury, a reloading injury will result in

activation of the immune system and recruitment of immune cells to the damaged region.

The presence of free radicals within the cell can initiate a pathway, likely through p38

and NFKB, leading to the production of IL-6 (119). Once produced, IL-6 can

chemotactically recruit immune cells to the site of production, i.e., the damaged muscle

fiber. Aiding in the chemotaxis of immune cells during reloading injury is the activation









of both classical and alternative complement (69). Complement is a series of reactions

that leads to the production of chemotactic moieties. Mitochondrial and cytoskeletal

proteins in the extracellular space, likely seeping out through sarcolemmal lesions, will

initiate the complement pathways. Further, complement inhibition will decrease immune

cell infiltration (69).

Despite the obvious infiltration of immune cells into fibers with injury resulting

from reloading, questions have been raised regarding the true nature of their role. Earlier

evidence has suggested they are deleterious particularly because they can generate a large

amount of oxidant damage within the cell. Frenette et al. (70) have constructed a time

course in which immune cell infiltration and the generation of tetanic force were

superimposed on each other. They were able to show that following 10 d of unweighting

and various durations of reloading, the greatest deficit in force came after only two hours

of reloading, where an increase in immune cell presence did not occur until 24 h

following reloading. This study indicates that the immune response is not responsible for

reductions in force following reloading. Instead, given the short duration of force

reduction, mechanical damage, oxidant damage, or E/C uncoupling is a far greater

likelihood. Indeed, E/C uncoupling has been noted after only two hours of reloading

(70). Additionally, both mechanical damage and/or oxidant damage could lead to Ca2

handling problems.

Oxidant Damage

During reloading the cell is subjected to a secondary oxidative stress, independent

of the oxidative stress experienced during immobilization, akin to an

ischemia/reperfusion type injury. Further, there is a loss of Ca2+ containment that occurs

within the cell during reloading potentially due to damage to the sarcolemma or via Ca2+









leak channels, although the precise mechanism is currently unknown (6, 96, 194). This

loss of homeostatic balance likely contributes to the secondary oxidative stress that

occurs during reloading in a similar fashion that occurs with unloading. The Ca2+

activated proteases, most likely calpain, convert xanthine dehydrogenase to xanthine

oxidase and result in the production of 02*- (149, 163, 214). Similar to unloading, the

iron catalyzed production of free radicals is also likely occurring (30).

Additionally, the increased intracellular Ca2+ concentration leads to an upregulation

of Ca2+ entering the mitochondria via the uniporter, resulting in a decreased membrane

potential (83, 98). With a reduction in membrane potential comes a reduction in the rate

of electron movement in the electron transport chain, increasing the possibility that

electrons will leak from the electron transport chain and produce 02'- (39, 58, 87).

Compounding matters is the reduction of MnSOD resulting from unloading, which is

responsible for the dismutasing of 02*- (115, 124). Elevated intracellular Ca2+

concentrations will also increase NOS activity and the production of NO*, which can

combine with 02 to form ONOO* (139). A positive feedback loop begins where

increased Ca2+ release propagates increased free radical production, and conversely, the

increased free radicals both increase Ca2+ release and inhibit Ca2+ clearance (2, 79, 80,

194, 223). Regardless of source, the cell experiences this secondary bout of oxidant

stress with a somewhat compromised antioxidant defense mechanism, making it

potentially more potent and deleterious.

While there were relatively few papers investigating oxidant stress and

immobilization, there are fewer still investigating oxidant stress and reloading. In one

investigation, reloading resulted in an increase in lipid oxidation as well as oxidized









glutathione. In a reloaded group also given the antioxidant, vitamin E, there was less

lipid oxidation and muscle mass was significantly larger when compared to reloaded

controls (110). In another investigation, SOD supplementation attenuated the reduction

in force associated with pliometric contractions both 10 min and three days after injury

indicating that losses in force were not solely mechanical in nature, but also had a

chemical or ROS mediated component (65, 242). Additionally, vitamin E

supplementation has been demonstrated to be of benefit as it has reduced creatine kinase

release and reduced oxidative damage (110, 148, 157). In contrast, other studies have

shown that vitamin E supplementation did not maintain torque, prevent Z-line streaming,

reduce creatine kinase release, or reduce macrophage infiltration following eccentric

damage nor was it beneficial to downhill running rats (15, 233).

Heat Shock Proteins

Overview

When the cell encounters a variety of stressful conditions a group of proteins called

stress shock proteins can be differentially expressed. Among the first characterized were

the heat shock proteins (hsp), which were first discovered to respond to a heat stress.

More recently, other stimulators have been identified including oxidative damage,

exercise, pharmacological agents, and UV light (for rev. 159). The hsps can be divided

into five families including hsp 90, 70, 60, 40, and the small hsps (shsp). These proteins

function to prevent protein degradation as well as assist in the elimination of proteins that

are too badly damaged for repair (236). The hsp 70 family member, hsp 72, and the shsp

family members, hsp 27 (hsp 27 in human is analogous to 25 in mouse and 28 in rats) and

hsp 32 (also commonly called Heme Oxygenase-1; HO-1) are of particular interest when









considering reloading as they are heat inducible and serve a variety of functions that may

be beneficial to a reloading muscle fiber.

Heat Shock Proteins 27, 32, and 72

Hsp 27 is by far the most common shsp with estimates as high as 3.4-ug/mg protein

in human biceps (220), while hsp 32 and 72 are readily inducible. Collectively, hsp 27,

32, and 72 have been shown to refold damaged or nascent proteins (chaperone) (16, 60),

interact with cytoskeletal components (181), help reduce oxidant stress (95, 109, 217),

and prevent apoptosis (16, 24, 73, 154, 155).

In its oligomeric form, hsp 27 functions to act as a chaperone in an ATP

independent manner. While in this conformation it can remove heat damaged proteins,

reduce protein aggregation, refold damaged proteins and can directly reduce ROS,

thereby reducing the potential for oxidative damage (193). Phosphorylation, occurring at

one of three serine residues, is one of the primary means of regulating hsp 27 activity

(94). The MAPK, p38, initiates a cascade that goes through MAPKAP 2/3 and

culminates with the phosphorylation of hsp 27 (82, 153). Upon phosphorylation, the

activated monomer will dissociate from the large oligomer and complete its job within

the cell (103, 193). As conditions become more stressful, hsp 27 becomes progressively

more phosphorylated (94). In its tri-phosphate form, hsp 27 exists in high levels as rod-

like tetramers (193). Currently, it is unknown what effect phosphorylation at the various

sites has on the role of this protein. Further, the precise role each of the conformations

this protein can take is also yet to be determined.

While hsp 27 is activated through a MAPK signaling pathway, hsp 72 is activated

by a more complex series of events. Under control conditions, hsp 72 is bound to heat

shock factor 1 (hsf-1) forming a dimer. Upon stress conditions, such as increased









intracellular protein damage, hsp 72 dissociates from hsf-1 and binds damaged proteins

(18). Hsf-1 then trimerizes and migrates to the nucleus where it binds the heat shock

element in the promoter region of the hsp 72 gene and facilitates transcription. Following

the repair of damaged proteins, hsp 72 is free in the cell and will once again bind hsf-1,

decreasing its ability to trimerize and produce more hsp 72 (13, 136). Alternatively, in

the heart, it appears as though stretch activated ion channels can lead to the activation of

hsf-1 and the subsequent increase in hsp 72 content (40). Further evidence of the

differing activation pathways of these two proteins is gained when glucocorticoids are

administered. Knowlton's group was able to demonstrate that hsp 72 was elevated along

with hsf-1, while hsp 27 remained at baseline levels (221).

Far more is known about hsp 72 in terms of function within the cell. Expanding the

role of hsp 72 already described is an involvement in protein synthesis and degradation.

This protein has been shown to modulate the rate of the elongation process, thereby

affecting protein synthesis (16, 122). It can also reduce or prevent protein aggregation

and appears likely that it can facilitate recognition of damaged proteins within aggregates

by partially unfolding them to allow ubiquitination (29, 129, 130, 204). Large protein

aggregates can inhibit proteasome activity and create a potentially cytotoxic

accumulation of damaged proteins (81). By reducing these aggregates, proteolytic

degradation can be maintained. Through a yet to be described mechanism, hsp 72 also

increased MnSOD and Bcl-2 levels within an I/R heart, which may provide an additional

mechanism of heat shock protein induced cardio-protection (222).

Hsp 32 is activated in a similar fashion as hsp 72. A heat shock element is found

in the promoter region of the rat hsp 32 gene (184). In addition, there are binding sites









for NFKB as well as activating protein-1 (AP-1), which is redox sensitive (123, 185). In

addition, IL-6 as well as a reduced GSH content will increase hsp 32 expression (191,

195). Furthermore, free heme and heat will induce an increase in hsp 32 expression (62,

63, 143, 184). Once induced, hsp 32 will degrade heme into three products including

CO, biliverdin, and iron. CO can have anti-inflammatory effects as it regulates

macrophage infiltration and cytokine release (28, 177). Further, CO can activate p38,

which will, in turn, activate a host of other protein kinases responsible for, among other

things, the activation of hsp 27 (24). Finally, CO has repeatedly been demonstrated to

reduce apoptosis (24). Biliverdin is a water-soluble antioxidant that is quickly converted

into the hydrophobic antioxidant, bilirubin.

The final product of hsp 32 activity is perhaps the most complicated. The release

of free iron within the cell raises the possibility that it will increase free radical synthesis

and hence, oxidant stress, as Fe2+ can participate in the Fenton reaction. Upon release of

iron into the cytosol, iron regulatory protein, which blocks ferritin translation as it is

bound to the mRNA, dissociates and ferritin is increased within the cell (61). Ferritin

binds free iron, making the elevation of free iron only transient. Further, when iron is

bound to ferritin in an oxidizing environment, such as during disuse or reloading, 02*-

can attack ferritin and cause the transient release of iron as the pro-oxidant Fe2+ (11, 195).

Despite the obvious risk of a transient increase of free iron within the cell, the summation

of these events is a reduction of free iron due to the increased ferritin (161, 178, 195).

Heat Shock Proteins, Oxidant Stress, and Potential as a Reloading Intervention

Among the many roles hsps can perform within the cell, their antioxidant properties

may be of most importance to a reloading skeletal muscle. Elevations in hsp 27 have

been demonstrated to provide protection against H202 as well as oxidant damage caused









by TNFa via NFKB (238). In rats with simulated Huntington's Disease, hsp 27 was

associated with higher survival rates and lower ROS production despite no reductions in

the expression of the Huntington protein, the cause ROS production (154, 237). Another

potent mode of cellular protection in the face of oxidant stress conferred by hsp 27 is the

reduction of GSSH to GSH (170).

The antioxidant effects of hsp 32 are primarily conferred through the release of

biliverdin and bilirubin from the degradation of heme, although the effects of ferritin are

also important in maintaining redox balance. Biliverdin and bilirubin have been found to

be as effective an antioxidant as vitamin E (217). It seems that they function through a

variety of means to reduce oxidant stress including acting as a peroxyl radical trap,

breaking the oxidant chain reaction in membranes, protecting against OH* attack, and

decreasing 02*- (166, 216). For example, both hsp 32 induction as well as bilirubin

supplementation were quite effective in protecting cells and reducing oxidant damage

following an ischemia/reoxygenation event (68). Further, infarct area and mitochondrial

damage were reduced in ischemia/reperfused hearts following either hsp 32 induction or

bilirubin exposure (45). In addition, free heme is pro-oxidant which indicates that its

removal from the cell is paramount.

In a similar fashion, hsp 72 has been shown to increase tolerance to oxidative

stress. Muscles from transgenic mice over-expressing hsp 72 showed higher force

generation following hypoxic fatigue, which the authors attributed to the protective effect

of hsp 72 to ROS damage (205). Further, cells transfected with an hsp 72 over-

expression gene demonstrated less cell death and damage when exposed to H202 (109).

More common; however, is an association between hsp 72 and a protective effect against









oxidant stress. It is worth noting that in many of these studies, hsp 27 and 32 were not

assessed and likely provided some sort of protective effect as well.

Untrained animals that are forced to exercise demonstrate higher oxidant stress, as

their antioxidant enzymes appear to be overwhelmed. Concomitantly, hsp 72 is over-

expressed in these animals following exercise, while their trained counterparts have

enzyme activity levels capable of handling the oxidant stress of one exercise bout and

show no elevation in hsp 72 expression. Heat stress has been shown to be protective

against an array of oxidative insults including skeletal muscle H202 exposure, skeletal

muscle ischemic injury, and liver carbon tetrachloride exposure (72, 165, 239).

As has been demonstrated previously, there is an increase in oxidant stress during

immobilization. The literature is inconsistent as it relates to changes in hsp 72 expression

with this type of intervention. It does appear though, that female rats demonstrate a

decline in expression, while the expression in male rats does not change (164, 201).

Regardless, there is not an increase in expression as may be expected during such a

stress. Taking advantage of what appears to be a biological oversight, two groups have

successfully used a heat intervention to attenuate muscle loss due to disuse atrophy (164,

201). In addition, oxidant stress was reduced in the group that was both immobilized and

heated when compared to one that was only immobilized (201).

Given that there is also a large increase in oxidant stress during reloading, it stands

to reason then, that providing a heat stress will likely result in elevated hsp expression as

well as decreased oxidant damage. As mentioned earlier, vitamin E supplementation has

been successfully used to reduce oxidant damage and enhance muscle regrowth following

immobilization (110). Further supporting the role of hsp' s in reloading skeletal muscle









are data that show that age matched transgenic mice over-expressing hsp 72 recovered

from a bout of pliometric contractions faster than wild-type adult and old mice (147).

These mice also did not suffer the well-characterized secondary drop in force production

occurring several days after injury. Further, adult transgenics also had a smaller damaged

muscle area than wild-type controls.

The summation of this body of evidence leads to the hypothesis that following

immobilization, heating will decrease oxidant damage to proteins and lipids during

reloading. Furthermore, it is anticipated that heat stress during reloading will result in

enhanced hypertrophy and higher IGF-1 pathway intermediate activation during

regrowth.














CHAPTER 3
METHODS

Design

All procedures and experiments were conducted with the approval of the

Institutional Animal Care and Use Committee at the University of Florida under the

animal use protocol designated D736. Animals were housed in a 12 hr light/dark

photoperiod in an environmentally controlled room. Upon arrival in the facility, animals

were handled daily for one week prior to the initiation of experiments in an effort to

minimize contact stress. Male Sprague-Dawley rats were randomly be divided into six

groups including a control group (Con) (n=10), a heated control group that will receive

multiple heat treatments on alternating days (ConH) (n=6), a group that is immobilized

for seven days (Im) (n=10), a group that is immobilized for seven days and receives a

heat treatment 24 hours before sacrifice (ImH) (n=6), a group immobilized for seven days

and allowed to reload for seven (R7C) (n=10), and a group immobilized for seven days

and allowed to reload for seven that receive heat treatments on alternating days during

reloading (R7H) (n=10; Figure 3-1).

Animals given heat therapy and immobilization will be heated 24 hours prior to

cast removal in order to assure that hsps are elevated when the casts are removed.

Animals assigned to the R7C and R7H will be allowed to reload for seven days before

sacrifice (Figure 3-2).

























Figure 3-1. Schematic of study design. Animals will be randomly assigned to one of six
groups. Control animals will be treated identically to other groups except that
they will not be immobilized. Animals not receiving a heat treatment will be
anesthetized identically to heated animals; however, the core temperature will
only be maintained rather than elevated to 41-41.5C as in heated animals.


All animals to Heat 7 day
receive heat are reloaders
heated


Mon Sun Mon Tues


All casts
All immobilized removed and
animals casted animals are
reloaded


Sac all 7 day
reloaders



Thur Sat Mon

Heat Heat


Sac Im and ImH
only animals

Figure 3-2. Schematic of protocol for one hypothetical week. Animals will be treated
such that representatives from multiple groups will be collected at the same
time. This potentates the probability that groups will be treated identically.

General Procedures

Immobilization and Reloading

Anesthesia was induced with a 5% isoflurane gas oxygen mixture and maintained

with a 1.5-2% isoflurane gas oxygen mixture administered through a calibrated air flow

meter (Veterinary Equipment and Technical Service, Gainesville, FL). Animals were









then immobilized bilaterally in the plantarflexed position as to cause maximal atrophy in

the triceps surae muscle group in accordance with the model described by Booth and

Kelso with modifications (22). Briefly, animals were wrapped in a protective adhesive

(MediporeTM Dress-it, 3M, St. Paul, Minnesota) so that the animals would not come in

contact with the plaster. The wrap begins in the supra-abdominal area, below the level

of the ribs, and continues down the abdomen of the animal and stops in the infra-

abdominal area and continues down the hind limbs. A quick drying plaster was then

applied and allowed to dry (Specialist, Johnson and Johnson, New Brunswick, New

Jersey). Finally a Plexiglas wrap was applied so that rats could not chew through the cast

(Scotchcast Plus, 3M, St. Paul, Minnesota).

In order to remove the casts, the animals were again anesthetized with isoflurane.

A rotor with a cutting wheel is used to remove the Plexiglas wrap. The casts were then

softened with warm water to make the plaster easier to cut. The cutting wheel was then

used again to remove the plaster cast. Animals were sacrificed or placed back into their

cages and allowed to return to normal activity for a period of seven days depending on

which group they were assigned.

Heat Treatment

Animals were anesthetized using isoflurane, as detailed above. A rectal probe was

inserted and secured to the tail to ensure that it would not become displaced (YSI, Yellow

Springs, OH). The animal was then wrapped in a pre-warmed thermal blanket (Kaz,

Hudson, NY) such that the tail and head were exposed. The tail was left exposed because

it serves as the anchor for the rectal probe; hence it cannot be curled into the blanket.

The head was visible enough to ensure that the nose cone was secure so that accidental

recovery from anesthesia would not occur.









Core temperature was continuously monitored and recorded every two minutes.

Heating time began as soon as the core temperature of the animal breached 41 C and

temperature was maintained at 41-41.50C for 30 min. At 30 min, the animal's core

temperature was lowered via convection cooling and continually monitored until the core

temperature was below 39.90C. Animals not receiving a heat treatment were treated

identically, except that core temperature was maintained for the 30 min period.

Animals receiving a heat treatment were heated 24 h prior to cast removal. We

have found that hsp expression peaks between 24 and 48 hrs following heating. Animals

allowed to reload for seven days will be heated 24 h prior to reloading and every other

day following until sacrifice.

Muscle Removal and Sample Preparation

On the day of sacrifice, a surgical plane of anesthesia was induced via

interperitonial pentobarbital injection. Then, the soleus was removed, trimmed of excess

fat, tendon, and nerve, blotted, weighed and immediately frozen in liquid nitrogen chilled

isopentane for subsequent analysis. To determine if muscle water content is altered, total

water content of muscles was determined by using a freeze drying technique

incorporating a vacuum pump with a negative pressure of-1 mm Hg. The measurement

was terminated when the same weight was recorded three times in succession during 48

hr interval.

Homogenization occurred following the technique of Solaro et al. (209). Briefly,

mass to buffer ratio was 20:1 such that if 0.03 g frozen tissue were utilized, it would

require 0.6 ml buffer. The resulting homogenate was then centrifuged at 300 g for 10

min to remove cellular debris and the supernatant removed while the pellet was









discarded. Protein concentration was then determined using the biuret technique of

Watters (234).

Biochemical Procedures

Western Blot

Samples were diluted to 1 mg/ml in sample buffer containing 62.5 mM Tris

(pH=6.8), 1.0% SDS, 0.01% bromophenol blue, 15.0% glycerol, and 5% 0-

mercaptoethanol. Samples were denatured via heating to 600C for 15 min in a glass bead

heater. Precisely 15-ug protein is loaded into 4-20% vertical precast gels (Cambrex,

Rockland, ME). Samples were then electrophoresed at room temperature for 30 minutes

at 50 v followed by 90 min at 120 v (BioRad, Hercules, CA). Gels were removed from

the electrophoresis apparatus and allowed to condition for 15 min in transfer buffer

containing 25 mM Tris, 192 mM Glycine, 0.02% SDS, and 20% methanol (pH=8.3).

Following the conditioning period, horizontal electrophoresis (100 v, 60 min, 40C) was

performed such that proteins were transferred to a nitrocellulose membrane with a pore

diameter of 0.2 um (BioRad, Hercules, CA). Alternatively, a dot blot was performed

such thatl5 ug of sample was added directly to the membrane and proceeds as a normal

western blot. Membranes were then washed in Tris-buffered saline containing .1%

Tween 20 (TTBS). Membranes were blocked by exposure to a 5% dehydrated milk

TTBS solution for 60 min. Membranes were washed for ten minutes, three times and

exposed to the appropriate primary antibody as follows: hsp 25 (SPA 801, Stressgen,

Victoria, British Columbia), hsp 72 (SPA 810, Stressgen), 4-hydroxy-2-nonenol (HNE;

HNE11 -S, Alpha Diagnostic International, San Antonia, Texas), nitrotyrosine (NT;

#9691, Cell Signaling Technology, Beverly, Massachusetts), AKT (#9272, CST),

phospho-AKT (#9271, CST), GSK3B (#9332, CST), phospho-GSK (#9331, CST),









p70S6K (#9202, CST), and phospho-p70S6K (#9205, CST) for 90 min. They were then

washed three times, for ten minutes and exposed to the appropriate secondary antibody

for 60 min (Amersham).

The secondary antibody was diluted in TTBS containing 1.5-2% milk protein.

Membranes were then washed for ten min, three times and exposed to ECL (Amersham,

Little Chalfont, Buckinghamshire, England) for two min. Finally, the membranes were

placed in a Kodak Image Station 440 CF developer and the emitted signal captured. The

signal was analyzed using the Kodak ID Image Analysis Software (Eastman Kodak

Scientific Imaging Systems, Rochester, NY).

Glutathione Peroxidase

All antioxidant enzymatic assays will be performed in triplicate in microplates

using a Spectramax 190 microplate reader (Molecular Devices, Downingtown,

Pennsylvania) using homogenate further diluted to 1:100 in PBS buffer.

GPX activity was determined by the method of Flohe and Gunzler (67) and was

based on the change in NADPH absorbance from the reaction catalyzed by GR. 240 ul

reaction cocktail containing .2974 U/ml GR, 1.25 mM GSH, and .1875 mM NADPH was

added to a microplate well followed by 30 ul homogenate. Following a three minute

incubation at 250C, 30 ul t-butyl hydroperoxide was added to the plate. The plate was

read every 15 sec for five minutes at 340 nm. A blank was run and will substitute the

homogenate for PBS. Activity was determined by first identifying the linear portion of

the resulting graph. Change/minute will be divided by 6.22 (extinction co-efficient for

NADPH) and multiplying by 2 (2 GSH: 1 NADPH). Activity was then multiplied by the

dilution factor and blank activity determined and subtracted from the total activity.









Glutathione Reductase

Glutathione reductase activity was determined by the method of Carlberg and

Mannervik (35). Briefly, a reaction cocktail was first prepared containing three solutions.

The first was 0.2 M PBS solution containing 2 mM EDTA. The second was 2mM

NADPH in 10 mM Tris-HCl (ph 7.0). The final solution comprising the reaction cocktail

was distilled water. The three were combined such that the mixture is 58.8% EDTA

solution, 5.9% NADPH solution and 35.3% water. They were mixed and 170 ul is added

to each well. Next, 20 ul sample was added to each well. The two were allowed to

incubate for 2 min at 300C and then 10 ul water (blanks) or GSSH solution (20 mM) was

added to the wells. Plates were read every 15 sec for three minutes at 340 nm. Activity

was calculated by subtracting the blank change/minute from the change/minute recorded

from each sample. Like before, it was divided by 6.22 to account for the extinction co-

efficient of NADPH and multiplied by 2 to account for the GSH:GSSH ratio. Finally,

this value was multiplied by the dilution factor.

Catalase

Catalase activity was determined by the method of Aebi (3). This assay is based on

changes in absorbance due to the degradation of H202. Sample was prepared as before

with the addition of several extra steps. Following centrifugation, ethanol was added in a

1:10 v/v ratio and incubated for 30 min. Next, 1% triton was added in 1:10 v/v ratio and

incubated for 15 min. This assay was run on plates designed for readings in the UV

range. Additionally, only one column was run at a time due to the rapid reaction rate.

Thirty-five microliters sample was loaded into each well. Next, 255 ul ofH202 solution

containing, 10 mM in 100 mM PBS (buffer for blanks), was then loaded into each well.

The reaction was immediately read for one minute with data being recorded every 5









seconds. Due to the abnormal kinetics of the catalase reaction, there is no defined unit

for catalase activity. Instead, activity was determined using a first order reaction rate

constant. The calculation was given by the equation:

K= (2.3/t) [loglO ((initial absorbance blank)/(final absorbance blank))

Activity = K*dilution factor

Superoxide Dismutase

Mn super-oxide dismutase (SOD), and CuZnSOD activity was determined

simultaneously by the method of McCord and Fridovich (150). Cytochrome C was

reduced by 02*-. The reduction of cytochrome C was slowed by the addition of SOD.

To perform the assay, 180 ul reaction cocktail containing 16.6mM purine and 88.8 uM

cytochrome c in 100mM PBS was added to each mircoplate well. Next, 15 ul

homogenate or buffer (blanks) is added followed by 20 ul KCn or buffer. The addition of

KCn will cause CuZnSOD not to function. Finally, 60 ul xanthine oxidase solution was

added containing 0.2% xanthine oxidase in PBS. Readings were taken every 15 sec for

four minutes at 550 nm. Activity was determined by the following calculation:

U/gww = [(blank change/minute homogenate change/minute)/(0.5 blank

change/minute)]* dilution factor

CuZnSOD activity = Total SOD MnSOD (with KCn).

Ferrous Oxidation Xylenol Orange

In order to determine the ferrous oxidation of xylenol orange (FOX), muscles must

be homogenized in methanol at a dilution of 1:10 in accordance with Hermes-Lima et al.

(86). Samples were homogenized in cold methanol at a 1:10 w/v ratio. Homogenates

were centrifuged at 1500 rpm for 10 min, and the supernated removed. The reaction

cocktail containing 75 ul 1 mM FeS04, 30 ul .25 M H2SO4, and 60 ul 1 mM Xylenol









Orange was prepared and added to the mircoplate well. Next, 129 ul water was added,

followed by 6 ul sample. These will be allowed to incubate for 24 h in the dark. They

will then be read in triplicate at 580 nm. The results will be plotted against a standard

curve constructed from cumene hydroperoxide. Total hyroperides are calculated as

follows:

mmol hydroperoxide/gr wet weight = (mmol concentration dilution factor)/grams

tissue.

ELISAS

Protein carbonyls were determined from the Zentech PC Test (Zenith Technology

Corp Ltd, Dunedin, NZ) (32). Hsp 32 content was measured using the Rat HO-1 ELISA

Kit (EKS 810, Stressgen).

Statistical Analyses

Data was compared using a one-way ANOVA in a predetermined comparison

involving the Con, Im, R7C, and R7H. Further, the ImH group will be used to show hsp

levels upon removal of the casts in animals that were heated and immobilized in

comparison to animals in the Im group. Finally, the ConH groups will be used to show

the effects of the heating protocol on hsp expression. ANOVAS that result in an F-test

that is significantly different will be compared using a Newman-Keuls post hoc test.

Alpha will be set at p<0.05.














CHAPTER 4
RESULTS

As mentioned earlier, two control groups were included in addition to the true

control. The first of these is identified as ConH and was treated like the Con except that

it was heated in a similar fashion as the R7H group. It was included to show that our

heating protocol would increase hsps and help to identify any variable that may respond

to our heating treatment. The second of the additional controls is the ImH group. This

group was treated like the R7H group, except that it was killed immediately prior to

reloading. The ImH group is included to determine if hsps are elevated upon reloading as

anticipated. Except where indicated, there was no difference between the Con and the

ConH and the Im and the ImH groups.

The presentation of the results begins with descriptive data from the whole animal

and the intact muscle, followed by indicators of oxidant stress and antioxidant enzyme

activities, culminating with data that may help to explain potential mechanism behind

these observations.

Whole Body Measures

Initial body mass did not differ between groups (Table 4-1). One week of

immobilization resulted in an approximate 10% weight loss for all immobilized groups.

During that same time period, the Con group increased body mass by approximately 7%.

Animals in the R7C and R7H groups were then allowed to reambulate for one week.

While this period did prevent further reductions in body mass, it did not result in an









increase in body mass. During the same time period, Con continued to increase body

mass by another 6%.

Table 4-1. Body weight changes at various time periods during this investigation.
Values are shown in grams and are presented as means SEM.
Con Im R7C R7H
Initial Body Mass (g) 357+3 357+3 3515 358+4
One Week Body Mass (g) 3816 3183 3126 317+5
Two Week Body Mass (g) 4059 NA 3146 301+4

All immobilized animals were match fed during this experiment. During the

unloading phase, there was no distinction in treatment between groups and as such, ad

libitum food consumption is similar across groups at 16 g/day. Beginning 24 hr prior to

reloading (day of first heating) animal feeding was matched to the R7H group. This

match was successful as both the R7C and the R7H ate approximately 15 g of food daily

for the week of reloading. Con rats were allowed to eat ad libitum for the duration of the

experiment and ate approximately 23 g of food daily.

Animals were allowed to drink water ad libitum during this experiment. Rats in the

Con group drank approximately 42 g water daily, which was significantly higher than all

other groups. During the immobilization phase of the experiment, animals drank

approximately 30 g water daily. This did not differ statistically from the 33 g water

consumed during the reloading phase of the experiment in either the R7C or the R7H

groups.

Whole Muscle Measures

Upon sacrifice of each animal, soleus wet weight was determined immediately

prior to freezing (Figure 4-1). Immobilization resulted in a 35% reduction in muscle

mass when compared to Con. While one week of reloading appeared to increase mass

when compared to the immobilized muscles, the increase of nearly 20% failed to reach









statistical significance. When reloading was combined with heat, the result was a

significantly larger muscle when compared to the muscles of the Im group. Furthermore,

the addition of heat to reloading caused significantly greater hypertrophy when compared

to reloading alone. Muscle water content was also determined and did not differ between

groups, indicating edema was not present in reloaded muscles at the time of sacrifice

(Table 4-2).


0.2-


t
-r--


0.1-




0.0 -
Con Im R7Con R7H

Figure 4-1. Wet muscle mass in control animals (Con), following one week of
immobilization (Im), immobilization followed by one week of reloading
(R7C), or immobilization followed by one week of reloading in combination
with a heat treatment (R7H). Data is presented as means + SEM. t
represents different from Con; j represents different from R7H.

When muscle mass was made relative to body mass (mg muscle mass/g body

mass), a similar pattern emerged (Table 4-2). Immobilization resulted in significant

atrophy when compared to Con. Reloading; however, returned this measure to Con

values. Heating in conjunction with reloading further augmented this measure, as it was

significantly greater than reloading alone and did not differ from Con.









Table 4-2. Relative muscle mass, water content, and wet weight to dry weight ratio
following one week of immobilization (Im), immobilization followed by one
week of reloading (R7C), or immobilization followed by one week of
reloading in combination with a heat treatment (R7H). Data is presented as
means SEM. t represents different from Con; j represents different from
R7H.
Con Im R7C R7H
Relative Muscle Mass (mg/g) 0.440.01 0.350.01t 0.410.02 0.480.03
% Water 78.20.5 77.50.4 78.10.5 78.50.7
Wet:Dry Ratio 4.610.12 4.450.08 4.590.10 4.700.15

Oxidative Damage

Oxidative damage was assessed in lipids by measurement of both total lipid

hydroperoxides (FOX) and lipid oxidation end products (HNE). Total lipid

hydroperoxides did not differ between groups; however the relative content of lipid

oxidation end products did differ between groups (Figure 4-2). In this measure,

immobilization increased HNE products when compared to Con. Reloading further

increased HNE products, as the R7C group was significantly higher than Con and Im.

Heating in conjunction with reloading returned HNE products to Con values.

In regard to protein oxidation, measures of toxic aldehyde and ketone attack

(protein carbonyls) as well as nitrosylated tyrosine residues (NT) were made. Protein

carbonyls did not differ between groups indicating that toxic aldehydes and/or ketones

may not be a significant cause of injury in this model at these time points. However, the

results of the NT dot blot assay were similar to the pattern of HNE change.

Immobilization resulted in a significant increase in damage that was still present during

reloading. Reloading in combination with heat eliminated this damage as NT was not

different between the R7H group and the Con group (Figure 4-3). These data were

confirmed by the conventional NT western blot, which revealed similar findings (Figure

4-3).










10.0-


tt#


a


Ifo & h i &


Con


Im R7C


R7H


Figure 4-2. 4-hydroxy-non-enol as determined by immuno-blotting following one week
of immobilization (Im), immobilization followed by one week of reloading
(R7C), or immobilization followed by one week of reloading in combination
with a heat treatment (R7H). Data is presented as means SEM. t
represents different from Con; j represents different from R7H; # represents
different from R7C.


7-. tt tt
n6-
,-1
04-
3-

2-


0 -9 -A -- -I -


Con


Im R7C


R7H


Figure 4-3. Nitrotyrosine as determined by dot blot immuno-blotting (top) and
conventional western blotting (bottom) following one week of immobilization
(Im), immobilization followed by one week of reloading (R7C), or
immobilization followed by one week of reloading in combination with a heat
treatment (R7H). Data is presented as means SEM. t represents different
from Con; j represents different from R7H.












2.- 1

S1.5-
0
^ 1.0-

S0.5-

0.0 -
Con Im R7C R7H

Figure 4-3. Continued

Anti-Oxidant Enzymes

As it is clear that the heat intervention does provide the cell with increased

resistance to oxidant damage it follows that this effect may be due to changes in

antioxidant enzyme activity. In this regard, both CuZn super oxide dismutase (SOD) and

MnSOD activities were measured along with catalase (Cat), and the glutathione handling

enzymes glutathione peroxidase (GPX) and glutathione reductase (GRX) (Table 4-3).

MnSOD, GPX, and GRX activities did not differ between groups. Immobilization

caused an increase in CuZnSOD activity, which was maintained during reloading.

Reloading combined with heat; however, lowered this measure to Con levels. Catalase

activity was significantly elevated with immobilization. Reloading lowered catalase

activity; however it was still elevated over Con values. Reloading with heat lowered

catalase activity to Con levels. These data suggest that heating does not increase

antioxidant protection by increasing antioxidant enzyme activity as in no instance was

R7H enzyme activity increased above that of R7C.









Table 4-3. Activities of various antioxidant enzymes following one week of
immobilization (Im), immobilization followed by one week of reloading
(R7C), or immobilization followed by one week of reloading in combination
with a heat treatment (R7H). Data is presented as means SEM. t
represents different from Con; j represents different from R7H; # represents
different from R7C.
Con Im R7C R7H
MnSOD (U/gww/min) 356+30 304+32 26141 28826
CuZnSOD (U/gww/min) 21218 31020t 29325t 22917
Catalase (U/gww/min) 2.200.07 2.670.04t# 2.440.09t 2.300.06
GPX (U/gww/min) 7.200.43 6.770.37 8.040.41 6.960.33
GRX (U/gww/min) 1159115 1137110 132888 113869

IGF-1 Pathway Activation

From the data above, it appears as though heating during reloading enhances

muscle regrowth following one week of immobilization. In order to determine if this

augmentation is due to IGF-1 pathway mediated protein synthesis, the protein content

and activation state of Akt and its downstream targets, Gsk and p70s6k, were determined.

Akt content was significantly reduced following one week of immobilization when

compared to all other groups (Figure 4-4). Reloading; however, resulted in a significant

elevation in Akt content when compared to all other groups. Interestingly, in conjunction

with heat, reloading returned Akt content to a level that was similar to Con. In a similar

fashion, Akt activation (as measured by Akt phoshorylation) was reduced following one

week of immobilization. Both reloading and reloading with heat resulted in a two fold

increase in Akt activation compared to Con. Since Akt content was higher in R7C than

R7H group, and pAkt was similar in both groups, it suggests that a greater proportion of

Akt is phosphorylated in the R7H group, which could indicate greater pathway activation

in this group.

In the ConH group, heating resulted in a significant reduction in Akt content as

levels fell to approximately 35% of Con. Surprisingly, this reduction was not found






44


when phospho Akt was assessed. This could further substantiate the notion that heating

increases IGF-1 pathway activation


Con Im R7C R7H


Figure 4-4. Akt content (top) and phosphor-Akt content (bottom) as determined by
immuno-blotting following one week of immobilization (Im), immobilization
followed by one week of reloading (R7C), or immobilization followed by one
week of reloading in combination with a heat treatment (R7H). Data is
presented as means + SEM. t represents different from Con; j represents
different from R7H; # indicates different from R7C; indicates different from
Im.

Immobilization did not reduce Gsk content nor change Gsk phosphorylation when

compared to Con (Figure 4-5). Reloading significantly increased Gsk content in the R7C

and R7H groups. Heat during reloading caused a significant increase in Gsk









phosphorylation when compared to all other groups, while reloading alone did not

increase Gsk phosphorylation state. Because reloading alone failed to increase Gsk

phosphorylation a breakdown in IGF-1 pathway signaling may be occurring. The lack of

change in phospho Gsk would decrease protein synthesis because a greater amount of

Gsk is present in the muscle in its active state. By comparison, in the R7H group the

increase in Gsk content is met by an increase in the phophorylation state meaning that

even though Gsk content is increased, it is being inhibited at a greater proportion than in

the R7C group, indicating a preservation of IGF-1 pathway signaling.


2.5-

2.0- -

S1.5-
0
1.0-

S0.5-

0.0 -
Con Im R7C R7H

Figure 4-5. Total Gsk (top) and phosphorylated Gsk (bottom) content as determined by
immuno-blotting following one week of immobilization (Im), immobilization
followed by one week of reloading (R7C), or immobilization followed by one
week of reloading in combination with a heat treatment (R7H). Data is
presented as means + SEM. t represents different from Con; j represents
different from R7H; # indicates different from R7C; indicates different from
Im.











2.5

2.0

S1.5-
0
S1.0-

S0.5-

0.0- ,
Con Im R7C R7H

Figure 4-5. Continued

P70s6k, a protein synthesis promoter, is downstream of Akt in the IGF-1 pathway

and is activated in response to an increase in Akt activation. One week of immobilization

failed to reduce p70s6k content; however, one week of reloading caused a significant

elevation in the expression of this protein when compared to both Con and Im, regardless

of heat (Figure 4-6). Furthermore, immobilization resulted in a significant reduction in

p70s6k activation. Like total protein, reloading and reloading with heat resulted in a

significant increase in activation of this protein. Additionally, heating resulted in

significant reductions in p70s6k content in the ConH group when compared to the Con

group and in the ImH group when compared to the Im group. Surprisingly, like Akt, the

reduction in p70s6k content did not affect phosphorylated p70s6k content in either the

ConH or the ImH groups as both were similar to the Con and Im groups, respectively.












S2.0- t_

o 1.5-
0
>% t #
^ 1.0-

S0.5-

0.0 -
Con Im R7C R7H


2- t*




0 1- -
(I1 I tI#




Con Im R7C R7H

Figure 4-6. Total p70s6k (top) and phosphorylated p70s6k (bottom) content as
determined by immuno-blotting following one week of immobilization (Im),
immobilization followed by one week of reloading (R7C), or immobilization
followed by one week of reloading in combination with a heat treatment
(R7H). Data is presented as means + SEM. t represents different from Con;
1 represents different from R7H; # indicates different from R7C; indicates
different from Im.

Heat Shock Proteins

As the antioxidant enzymes did not cause the decrease in oxidant damage found in

this investigation, it is reasonable to suggest that increases in hsps may have played a

role. Hsp 27 was significantly reduced following immobilization when compared to all

other groups (Figure 4-7). Following one week of reloading, hsp 27 levels increased to

above Con levels. Application of a heat treatment further elevated hsp 27 levels by an









additional 25%. Hsp 27 was similar between the Con and ConH and the Im and ImH

groups. Hsp 72 followed a very similar pattern when compared to hsp 27 in that there

was a significant reduction in hsp 72 levels following immobilization (Figure 4-8). This

reduction appears to be offset by reloading, as R7C did not differ from Con. Heating

during reloading increased hsp 72 by an additional 30%. Hsp 72 content was nearly

doubled in the ConH compared to the Con group and increased by 75% in the ImH group

compared to the Im group. Hsp 32 behaved in a manner that differed greatly from the

other hsps (Figure 4-9). Immobilization actually increased hsp 32 content by 32%, while

reloading returned hsp 32 content to levels that were similar to Con. Reloading with heat

increased hsp 32 levels by 37% when compared to Con and 25% when compared to R7C,

but did not differ from Im. Surprisingly, heating did not increase hsp 32 content in the

ConH group when compared to the Con group. Heating resulted in a 25% increase

(p=0.06) in the ImH group when compared to the already elevated Im group. The content

of all hsps in R7H was significantly higher than R7C.


2-

t t
I t --r-




0a t #




Con Im R7con R7H

Figure 4-7. Relative hsp 27 content following one week of immobilization (Im),
immobilization followed by one week of reloading (R7C), or immobilization
followed by one week of reloading in combination with a heat treatment
(R7H). Data is presented as means + SEM. t represents different from Con;
1 represents different from R7H; # represents different from R7C.











1.5-


: 1.0-
0
O
>.
.; 0.5-


0.0-


IL j 7I


Con


Im R7C


R7H


Figure 4-8. Relative hsp 72 content following one week of immobilization (Im),
immobilization followed by one week of reloading (R7C), or immobilization
followed by one week of reloading in combination with a heat treatment
(R7H). Data is presented as means SEM. t represents different from Con;
1 represents different from R7H; # represents different from R7C.


Con
Con


Im R7C


R7H


Figure 4-9. Hsp 32 (HO-1) content following one week of immobilization (Im),
immobilization followed by one week of reloading (R7C), or immobilization
followed by one week of reloading in combination with a heat treatment
(R7H). Data is presented as means SEM. t represents different from Con;
1 represents different from R7H; # represents different from R7C.


I:














CHAPTER 5
DISCUSSION

This investigation sought to establish that providing a heat intervention during

muscle reloading following one week of immobilization would augment muscle

regrowth. To that end, four hypotheses were tested including: (1) Heat treatment will

cause an increased muscle regrowth rate following eight days of reloading; (2) Heat

treatment will reduce oxidative damage to both lipids and proteins during reloading; (3)

Heat treatment will result in a reduction in antioxidant enzyme activity during reloading;

(4) Heating will cause an increased activation of IGF-1 pathway intermediates during

reloading. This is the first investigation demonstrating that heating will reduce

oxidative damage during reloading and accelerate muscle regrowth. It was also

demonstrated that the antioxidant protection was not due to antioxidant enzymes, which

further implicate heat shock proteins as responsible for the observed antioxidant effect.

Lastly, this is the first investigation to show that heating does affect IGF-1 pathway

signaling during reloading, which may help to explain why muscle regrowth occurred at a

higher rate in the R7H group when compared to the R7C group.

Muscle Mass

In support of the first hypothesis, heat treatment did augment muscle regrowth

following one week of immobilization. Soleus muscles in the R7H group were

significantly larger, both absolutely and when normalized for body weight, than soleus

muscles in the R7C group. Potential support for this finding can be found from a recent

study by Goto et al (76). In this investigation, there does not appear to be a difference in









soleus wet weight between a reloaded and a reloaded heated group. However, when

normalized for body weight, it does appear that the heated reloaded group is

approximately 15% larger following seven days of reloading despite the fact that there is

also no difference in body weight. Resolution for this apparent discrepancy could be that

in the Goto study (76), only one heating bout was given during the 10 day reloading

phase. Furthermore, they report only that hsp 72 is elevated while ignoring other heat

shock proteins that may also be important in muscle regrowth augmentation including

hsp 27 and 32. It is quite possible that both of these proteins may have fallen to control

levels during the ten days of recovery following the lone heating bout. As the protective

effect was minimal, if at all present, it seems important to consider that elevations of

other or additional proteins beside hsp 72 may be required in order to maximize the

potential of heat therapy in skeletal muscle regrowth augmentation.

Several other studies have investigated the effect of heat on hypertrophy or cell

culture growth. Seven days following a single heat stress (410C for 60 min) soleus

muscles were larger when compared to a control group (106). Another study, using a

similar design (41-42C for 30 min) showed an increase in soleus wet mass normalized

for body weight as well as dry muscle weight in a heated group when compared to a

control group (230). Lastly, it has been shown that heat increased protein content in rat

skeletal muscle cells (L6) when compared to a control (77).

Only one investigation has attempted to augment muscle regrowth following disuse

using an antioxidant (110). In that study, the degree of atrophy after one week of

reloading in the reloaded control group was approximately 45%, while the vitamin E

supplemented reloaded group was approximately 30%. Stated in these terms, the degree









of atrophy in this investigation in R7C was approximately 70%, while the degree of

atrophy in R7H group was approximately 40%. This indicates that heating performed at

least as well in assisting muscle regrowth as exogenous vitamin E.

In another investigation, animals were supplemented with torbafylline in an attempt

to decrease muscle atrophy during 5 wks of immobilization and enhance muscle regrowth

during four weeks of reloading (1). Torbafylline is a xanthine derivative with a yet to be

defined mechanism of action. In various conditions, it has been reported to function as

an antioxidant, regulator of inflammation, and inhibitor of the ubiquitin proteasome

pathway (1, 46). It failed to protect muscle mass during immobilization and was not

effective as an agent to enhance hypertrophy during regrowth. It may be the reason it

seemed so unsuccessful was that the time of immobilization was quite long at five weeks

and the time of reloading was also quite long at two and four weeks. Any beneficial

effect could have well been overcome by the severe state of atrophy and missed during

reloading due to the prolonged time period of regrowth. Torbafylline was able to return

mitochondrial density to control values and enhanced fatigue resistance during the

reloading period.

Oxidative Stress and Antioxidant Enzymes

In the present study, heating significantly reduced oxidative stress caused by

reloading in both lipids and proteins, in support of hypothesis 2. Furthermore, this

reduction returned oxidative stress markers to control levels indicating that heating is a

powerful antioxidant. In another study, it was shown that antioxidant supplementation

both enhanced muscle regrowth and reduced oxidant damage following immobilization

(110).









In a previous investigation, using a similar heating paradigm, we have shown that

heating can reduce oxidative damage during immobilization (as well as attenuate muscle

atrophy) (201). Further, other investigations have demonstrated that heating can be a

potent antioxidant intervention in a host of free radical generating conditions. In one

investigation evaluating heart function and infarct size following an IR injury, heated

hearts performed significantly better and had smaller infarcts sizes when compared to

hearts that were not heated (100). In several studies meant to simulate peripheral

vascular disease, IR injury of the limb was reduced with heating (72, 165).

In this investigation, lipid oxidation was measured by assessing the ferrous

oxidation of xylenol orange as well as the total HNE products. HNE was significantly

increased in the Im group as well as the R7C group when compared to both Con and

R7H, while the results of the FOX assay remained unchanged, regardless of treatment

group. While they both are used as indices of lipid oxidation, what they measure is

subtly different. The FOX assay measures total lipid hydroperoxides, likely from

polyunsaturated fatty acid oxidation; however, HNE is a measure of lipid peroxidation

end products that have reacted with proteins and left a characteristic fingerprint (183).

Furthermore, the FOX assay, while a vast improvement over the TBARS assay, is still

quite a bit more variable than the more stable HNE measure. The fact that one is

different while the other remains unchanged should not suggest that the increase in lipid

oxidation is tenuous, but that total hydroperoxides were not different; however

breakdown products were elevated in the Im and R7C groups when compared to the Con

and R7H groups.









Analysis of the protein oxidation data is similar to that of the lipid oxidation data in

that one measure was able to detect a difference, while another was not. The measure of

protein carbonyls is a very precise assay that allows the quantification of proteins that

have been attacked by toxic aldehydes or ketones. Nitrotyrosine, on the other hand, is a

measure of nitrosylated tyrosine residues as a result of NO* or ONOO* attack. These

data may indicate that NOS dysfunction may play a more critical role in free radical

generation in this model than previously thought as both NO* and ONOO* ultimately

originate from NOS. That one measure was different while the other remained

unchanged should not suggest that the increase in protein oxidation is tenuous, but that

protein carbonyls were not increased; however, nitrosylated tyrosine residues were

elevated in the Im and R7C groups when compared to the Con and R7H groups.

Because heating reduced oxidant damage, it becomes important to assess

antioxidant enzyme status. In this regard, CuZnSOD and MnSOD, catalase, glutathione

peroxidase, and glutathione reductase activities were measured. As there is no

information regarding antioxidant enzyme activity during reloading in the literature, our

hypothesis regarding reduced antioxidant enzyme activities in the R7H group when

compared with the R7C group is based on the notion that there is a strong record of

oxidant stress associated with reloading and pliometrically damaged skeletal muscle. In

many instances, increases in oxidant stress are countered by increases in antioxidant

enzyme activities.

MnSOD, GPX, and GRX activities did not change in response to immobilization or

reloading in conjunction with what has been found previously (111, 115, 201). The

possible exception to this is GRX activity, which, in separate investigations, did not









change in response to immobilization and increased in response to immobilization (111,

115, 201). Possible resolution to the incongruent findings, is that different rat strains

used in these investigations produced dissimilar results. Furthermore, MnSOD activity

was not increased in response to immobilization or reloading, which could indicate that

mitochondrial production of 02*- is not elevated in these conditions (12, 84).

From our data, we were able to show that CuZnSOD activity was increased in

response to immobilization and remained elevated during reloading; however, reloading

in combination with heat reduced CuZnSOD activity compared to control. This is in

good agreement to what has been found previously during immobilization or unloading

(111, 115, 124, 201). Because CuZnSOD activity is induced in response to 02*-, it may

indicate elevations in 02*- content in the cytosol of Im and R7C animals (12, 84).

Moreover, it may also indicate that there is a reduction in 02*- in R7H animals when

compared to the Im and R7C animals. In addition, immobilization and reloading resulted

in an elevated catalase activity when compared to control, while reloading in combination

with heat was similar to control. This is in good agreement with what has been found

previously during immobilization and unloading (111, 115, 201). Both of these instances

demonstrate support for hypothesis three in that heating during reloading resulted in a

reduction in antioxidant enzyme activity. As antioxidant enzyme activities were reduced

in the R7H group when compared to the R7C group, and oxidant damage, and

presumably 02*- content, were reduced in the R7H group when compared to the R7C

group, alternative antioxidant moieties must be considered (12, 84).

Heat Shock Proteins

Changes found in this investigation involving hsp expression following either Im or

reloading appear to be typical of what has been found previously. Hsp 72 is by far the









most studied of the hsps, and as such, provides the most data for comparison. Following

unloading or immobilization, most studies show a reduction in either hsp 72 mRNA

content or protein expression (19, 164, 215) while others have detected no change (172,

201). A previous suggestion was that the gender of the animal was the primary

determinant in either a reduction or no change in hsp 72 mRNA content/ protein

expression as only one study involving male rats was able to detect a reduction in hsp 72

expression (173). As this work represents the second investigation using males to detect

a reduction in hsp 72 expression, this notion should be reconsidered.

Reloading resulted in a sharp increase in hsp 72 expression following the reduction

seen during immobilization in this data set. An increase in hsp 72 expression during

reloading has been found previously (76, 173). Furthermore, reloading resulted in a four-

fold increase in hsp 72 mRNA in only four hours of ambulatory recovery (19). In the

present study, heating in combination with reloading further increased hsp 72 levels to

that above reloading alone. It appears as though a similar result was found in another

study (76).

Hsp 27 has been largely overlooked in the unloading/reloading literature. In this

investigation, immobilization resulted in a reduction in hsp 27 expression, while

reloading resulted in a dramatic increase, to levels above Con. Heating combined with

reloading increased hsp 27 expression further than reloading alone. As this protein has

been largely ignored with regard to unloading/reloading, it is difficult to interpret this

finding.

In regard to unloading/reloading work, hsp 32, like hsp 27, has also been

overlooked. In this investigation, immobilization resulted in a large increase in hsp 32









expression. Previous work supports the notion of an increase in hsp 32 expression

following disuse (93). As hsp 32 is increased in response to free heme, it may indicate

that myoglobin is being degraded due to free radical attack (142, 143). Furthermore,

there is evidence that during periods of oxidant stress, additional sources of heme may be

available. These include cytochrome C, newly synthesized heme, and hemoglobin (142,

145). Newly synthesized heme is particularly vulnerable to catabolism by hsp 32 because

during periods of oxidant stress the reduction in protein synthesis may mean that the

macromolecule it was going to join will no longer be synthesized leaving it available for

hsp 32 activity (142). Lastly, there is also a likelihood that with lipid oxidation, as in this

model, hemoglobin can leak into the cell and ultimately contribute to the free heme pool

(144, 146). With reloading, hsp 32 content was returned to baseline, while hsp 32

content in the R7H group reached that of Im. Given the similarities between

immobilization and reloading in terms of oxidant stress, it is surprising that in one

condition hsp 32 is induced, while in the other it is at control levels. It may be that in the

environment of an atrophying muscle, the availability of newly synthesized heme is quite

large (142, 145). By comparison, the environment of a reloading muscle is characterized

by increases in protein synthesis that may reduce the free heme pool.

As the antioxidant enzymes were not elevated in the R7H group and the oxidant

damage was lower in this group, alternative antioxidant substances must be considered.

Implicit throughout this project is the notion that heat shock proteins convey the

antioxidant protection found through heating. Importantly, hsps were increased in each

case in the R7H group when compared to the R7C group indicating that they may be

responsible for the observed antioxidant effect. The antioxidant capabilities of many of









these proteins are quite extensive. For example, in cells over-expressing either hsp 27 or

72, cell survivability and damage was significantly less when compared to control cell

types (109, 238). Indeed, animals over-expressing hsp 72 recovered force significantly

faster following a lengthening contraction protocol than did wild-type animals (147).

Bilirubin, a product of hsp 32 activity, is a potent antioxidant and, like hsp 27 and 72,

will prevent cell death and damage when an oxidant stress is encountered (68).

Furthermore, gene array studies have shown that the only discernable trend

following heating is an increase in heat shock proteins. Comparatively, these studies

demonstrate inconsistent changes in other antioxidant substances including MnSOD,

CuZnSOD, catalase, and various glutathione handling enzymes (192, 210, 211, 243). For

example, in one investigation glutathione-S-transferase and MnSOD are reduced, while

in another, both are increased following heating (192, 243). Nevertheless, the possibility

remains that proteins other than hsp 27, 32, or 72 are responsible for the observed

antioxidant effect.

IGF-1 Pathway

Muscle growth in the soleus following disuse atrophy appears to occur in two

phases. The first, lasting approximately seven days, is dependant upon the IGF-1

pathway, while growth thereafter is reported to be dependant upon calcineurin and

satellite cells (20, 158). As the regrowth period in this investigation was limited to one

week, IGF-1 pathway activation was assessed. In this pathway, IGF-1 leads to the

activation of three parallel pathways mediated by Akt. Down stream of Akt, Gsk

phosphorylation decreases its inhibitory role on protein synthesis and mTOR activation

results in the phosphorylation of p70s6k, which increases its protein synthesis role. In









order to evaluate IGF-1 pathway activation, total Akt and phospho-Akt, total p70s6k and

phospho-p70s6k, and total Gsk and phospho-Gsk were measured.

Immobilization resulted in a reduction in total Akt followed by an increase in total

Akt to that of nearly two fold Con in the R7C group. In another investigation, 10 days of

unweighting resulted in a trend toward a reduction Akt content. During reloading, total

Akt content was increased, like in the present investigation (43). Heating appeared to

blunt the increase during reloading, as total Akt was increased to Con levels, but not

beyond. Phospho-Akt content followed a similar pattern to that of total in that there was

a reduction during the immobilization phase that was met by a strong induction in the

R7C and R7H groups that did not differ. That phospho-Akt was down with

immobilization and increased in response to reloading are in good agreement with other

studies of similar design (43, 219). As total Akt was increased in the R7C group more

than in the R7H group and the phospho-Akt content did not differ, a greater proportion of

Akt must have been phosphorylated in the R7H group, thus, an increase in IGF-1

pathway activation could be indicated. Previous work has shown an increased Akt

activation with heat shock, likely mediated through hsp 32 and 72, but not hsp 27 (64,

118, 202). Furthermore, hsps 32 and 72 have maintained Akt activity in the face of

oxidant stress (64).

P70s6k content was reduced with immobilization and increased similarly in the R7C

and R7H groups. P70 s6k phosphorylation followed a similar pattern as total content;

however, immobilization did not cause a change in p70s6k phosphorylation while it was

increased similarly in the R7C and the R7H groups. In a similar investigation, unloading

did not reduce phospho-p70s6k content, and reloading caused a significant increase in









phospho-p706k content indicating that the present study and that mentioned are in good

agreement (219). It does appear though, that the increase in p70s6k content is

approximately 20% larger in the R7C group when compared to the R7H group. Further,

the phosphorylation of p70s6k is approximately 20% greater in the R7H group when

compared to the R7C group. While these differences are not statistically significant,

these findings potentially support the notion that there is increased IGF-1 pathway

activation in the R7H group when compared to the R7C group.

This idea is further supported by the analysis of the Gsk and phospho-Gsk data.

Immobilization did not cause a reduction in total Gsk content; however, reloading caused

a nearly two fold increase in the R7C and R7H groups. These findings are in good

agreement with previous work from Booth's lab using a study of similar design (43).

Like total Gsk, phospho-Gsk did not change in response to immobilization. Similar

results have been found previously (43). Phospho-Gsk was also not increased during

reloading; however, reloading in combination with heat resulted in a two-fold increase in

phospho-Gsk content. In a previous investigation, Childs et al. (43) showed that

reloading resulted in an increase in Gsk phosphorylation. Resolution to this apparent

discrepancy remains elusive. As the present investigation represents only the second

such study, and such divergent results were found, additional testing of this variable

should be considered. In another investigation, heat shock increased Gsk

phosphorylation during oxidant stress compared to non-heated cells (202).

These findings support the premise that heating, and perhaps hsps specifically,

reversed IGF-1 pathway dysfunction seen with reloading. This is particularly true in light

of evidence that IGF-1 pathway activation has shown to be preserved by heat stress or









hsp induction in the face of oxidant stress, as in the case with reloading (64, 118, 202).

Furthermore, because muscle regrowth is IGF-1 dependant during this phase of

hypertrophy, these data may contribute toward an understanding of why the muscle mass

in the R7H group was significantly larger than the muscles in the R7C group.

Integration

In this investigation, the global hypothesis tested was that heating will enhance

muscle growth following immobilization. Ultimately, this is based on results from

Kondo et al. (110) who were able to use vitamin E to increase the rate of regrowth in a

similar study design. That study also showed that there is an increased amount of oxidant

damage with reloading that is attenuated with vitamin E supplementation. These authors

conclude that a component of muscle regrowth is inhibited by oxidant stress and that

exogenous antioxidants will provide some help. We, and others, have shown that various

hsps, and heating in general, can act as an antioxidant, thus framing the initial belief in

this project (72, 164, 201, 238, 239). The notion that heating, or heat mediated induction

of hsps, could enhance muscle regrowth was further strengthened by the work of

McArdle et al. (147) who were able to show that rats over-expressing hsp 72 recovered at

a faster rate than wild type rats following an injury protocol that is similar to what is

experienced during reloading.

That muscle weights were larger in heated reloaded rats compared to reloaded rats

supports the hypothesis that heating augments muscle regrowth. Furthermore, heating

acted as a potent antioxidant in this model, returning lipid oxidation and protein oxidation

to control levels. This further supports the notion that heating acted as an antioxidant and

as such increased the rate of hypertrophy in a similar fashion to the work of Kondo et al.

(110). Assessment of the antioxidant enzyme activities revealed that they are not









responsible for the antioxidant protection; hence, alternative sources must be sought. We

have previously found similar results in an immobilization study that used heat as a

countermeasure to oxidant stress and muscle atrophy (201). It is speculated that hsps 27,

32, and 72 are conferring the antioxidant effect seen in the heated reloaded group. The

design of this study prohibits stating conclusively that these proteins are responsible.

Nevertheless, these proteins are increased in expression in the R7H group compared to

the R7C group and can act as antioxidants. Furthermore, analysis of gene array studies

reveals inconsistent changes in many other antioxidant substances (192, 210, 211, 243).

The study design does allow for the conclusive statement that heating is an antioxidant

intervention, independent of antioxidant enzymes, and that reduction of oxidant damage

results in enhanced skeletal muscle regrowth.

The last portion of this study dealt with the possibility that heating may enhance

regrowth by affecting the activation of the IGF-1 pathway, possibly due to hsp interaction

with IGF-1 pathway proteins. Previous work has shown that hsp 32 and 72 will increase

Akt activity as well as increase Gsk phosphorylation during exposure to oxidant stress

such as is the case with reloading (64, 118, 202). Akt comes early in the IGF-1 pathway

and the results indicate that there is an increased activation of this protein in the R7H

group compared to R7C as suggested by previous work (64, 118, 202). Down stream of

Akt are both p70 s6k and Gsk in divergent pathways. Gsk is directly down stream of Akt

and the results clearly show an increased phosphorylation of this protein, leading to an

inhibition of its anti-synthesis function in the R7H group compared to the R7C group as

has been found previously (202). P70s6k also lies down stream of Akt (Akt/mTOR/p70
s6k) and shows the familiar trend of an increased activation in the R7H group compared to









the R7C group. It could be that heating acts to reduce oxidant stress, which in turn helps

to maintain IGF-1 pathway fidelity, or that the reduction in oxidative stress seen with

heating and the increased IGF-1 activation are independent events contributing to the

same ultimate outcome increased muscle mass in a heated reloaded group when

compared to a group that is reloaded without intervention.

In summary, we have shown that heating will increase the rate of hypertrophy

following one week of immobilization. Further, we are the first to demonstrate that

heating will reduce oxidant damage in this model, in an antioxidant enzyme independent

fashion. Lastly, we show that heating during reloading will help to maintain IGF-1

pathway activity. We suggest that hsps 27, 32, and 72 are likely responsible for the

reduction in oxidant damage as they are induced in the heated reloaded group while

inconsistent changes have been found for various other antioxidants following a heat

stress. Additionally, we believe it possible that hsps 32 and 72 may be interacting with

IGF-1 pathway intermediates to promote increased pathway stability. The culmination of

reduced oxidant damage and increased IGF-1 activity likely caused the increased muscle

mass found in the reloaded heated group when compared to reloading alone.















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BIOGRAPHICAL SKETCH

Joshua Taylor Selsby grew up in Dayton and rural central Ohio before graduating

from Mt. Gilead High School in 1995. He then attended the College of Wooster for his

undergraduate education where he was a member of the men's swimming team for four

years. During his time there he completed a thesis that combined his love of science with

love of swimming entitled "Swim Performance following Creatine Supplementation in

Division III Athletes" and was advised by Drs. Michael Kern and Keith Beckett. He

graduated in May 1999 with a BA. Following graduation, he attended The Ohio State

University in the Exercise Physiology Department under the mentorship of Dr. Steven

Devor. His thesis was titled "A Novel Mg-creatine Chelate and a low Dose Creatine

Supplementation Regimen Improve Work." After graduating in June 2001 with an MA

he attended the University of Florida under the supervision of Dr. Stephen Dodd in the

Muscle Physiology Lab. While at UF he had the opportunity to be involved in several

different projects before developing a keen interest in heat shock proteins. His thesis was

titled "Does Heat Treatment Facilitate Muscle Regrowth following Hind Limb

Immobilization." Upon defense of his dissertation, Joshuajoined the lab of Dr. Lee

Sweeney at the University of Pennsylvania as a post-doc so that he could continue to

develop as a scientist and learn new techniques to better explore skeletal muscle

adaptation.