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Trolox supplementation during mechanical ventilation attenuates contractile dysfunction and protein degradation

University of Florida Institutional Repository

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TROLOX SUPPLEMENTATION DURING MECHANICAL VENTILATION ATTENUATES CONTRACTILE DYSFUNC TION AND PROTEIN DEGRADATION BY JENNA LEIGH JONES BETTERS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN EXERCISE AND SPORT SCIENCES UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Jenna Leigh Jones Betters

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This thesis is dedicated to my husband, Chad Betters, and my parents, Jim and Sue Jones, for their love and support.

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iv ACKNOWLEDGMENTS This project would not be completed w ithout the support and assistance of many people. I would like to thank Dr. David Cris well, my committee chai r and mentor, for his time and assistance with this th esis. Also, Dr. Scott Powers and Dr. Steve Dodd served as advisors to me during this process. I would es pecially like to thank Dr. Powers for the use of his laboratory equipment to complete this study. Dr. R. Andrew Shanely, Darin van Ga mmeren, Darin Falk, and Dr. Keith DeRuisseau devoted their time and talents to co mpleting this project. I thank them for all of the early mornings, as well as the late ni ghts. I also thank Tossaporn Yimlamai for his assistance with measuring the proteasome activity. Lastly, I thank my husband, Chad Bette rs, for helping me through the tough moments, and my parents, Jim and Sue Jones, for encouraging me to persevere through challenges.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... ix CHAPTER 1 INTRODUCTION........................................................................................................1 Background...................................................................................................................1 Significance of the Study..............................................................................................6 2 LITERATURE REVIEW.............................................................................................8 Skeletal Muscle Adaptations to Unloading..................................................................8 Oxidative Stress and Skeletal Muscle.........................................................................19 Skeletal Muscle Unloading and Protein Degradation.................................................21 Antioxidant Supplementati on and Skeletal Muscle....................................................23 Summary.....................................................................................................................27 3 METHODS.................................................................................................................28 Experimental Design..................................................................................................28 Diaphragm Contractile Function................................................................................30 Protein Degradation....................................................................................................32 20S Proteasome Activity............................................................................................33 Total and Non-protein Thiols.....................................................................................33 Statistical Analysis......................................................................................................35 4 RESULTS...................................................................................................................36 Systemic and Biologic Responses to Treatment.........................................................36 Effects of Anesthesia on Dia phragm Contractile Properties......................................36 Effects of Mechanical Ventilat ion on Contractile Properties.....................................36 Effects of Trolox on Contractile Properties................................................................37 Protein Degradation....................................................................................................38

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vi 20S Proteasome Activity............................................................................................38 Oxidative Stress..........................................................................................................38 5 DISCUSSION.............................................................................................................48 Trolox Attenuates Mechanical Ventilati on-induced Contractile Dysfunction and Proteolysis in the Rat Diaphragm:Introduction.....................................................48 Materials and Methods...............................................................................................49 Results........................................................................................................................ .55 Discussion...................................................................................................................57 LIST OF REFERENCES...................................................................................................63 BIOGRAPHICAL SKETCH.............................................................................................68

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vii LIST OF TABLES Table page 4-1 Body and Diaphragm Weights of C ontrol, Spontaneously Breathing, and Mechanically Ventilated Animals............................................................................39 4-2 Maximal Isometric Twitch and Tetani c Force of Control, Spontaneously Breathing, and Mechanically Ventilated Animals...................................................39 4-3 Contractile Parameters of Maximal Isometric Twitch and Tetanic Forces of Control, Spontaneously Breathing, and Mechanically Ventilated Animals.............40

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viii LIST OF FIGURES Figure page 4-1 Force-frequency responses.......................................................................................41 4-2 Responses of in vitro diaphragm strips to a 30-min fatigue protocol......................42 4-3 Percent of initial force maintained by in vitro diaphragm strips after a 30-min fatigue protocol........................................................................................................43 4-4 Total in vitro diaphragmatic protein degradati on as measured by the rate of tyrosine release during a 2-hour incubation.............................................................44 4-5 Chymotrypsin-like activity of the 20 S proteasome in diaphragm tissue.................45 4-6 Total thiol concentration in diaphragm tissue..........................................................46 4-7 Non-protein thiol concentr ation in diaphragm tissue...............................................47

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ix Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science in Exercise and Sport Sciences TROLOX SUPPLEMENTATION DURING MECHANICAL VENTILATION ATTENUATES CONTRACTILE DYSFUNCTION AND PROTEIN DEGRADATION By Jenna Leigh Jones Betters May 2004 Chair: David Criswell Major Department: Exercise and Sport Sciences Prolonged, controlled mechanical ventil ation (MV) results in diaphragmatic atrophy and reduced diaphragmatic force gene rating ability. To investigate whether an antioxidant, Trolox, could attenu ate atrophy and force loss, we tested the hypothesis that Trolox supplementation during MV would reduce protein de gradation and contractile impairments of the diaphragm by preventing oxidative damage. Further, we postulated that proteolysis during MV is mediated by the ATP-ubiquitin-dependent proteasomal pathway. Sprague-Dawley rats were anesthe tized, tracheostomized, and mechanically ventilated with 21% O2 for 12 hours. Trolox was intrave nously infused in a subset of ventilated animals. These were compared to groups of spontaneously breathing (SB) animals anesthetized for 12 hours, as well as an acutely anesthetized control group. Twelve hours of MV resulted in a 17% decrease in maximal tetanic force compared to

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x controls. However, Trolox supplementation duri ng MV completely attenuated the loss of maximal force. Proteolysis, measured as the release of free tyrosine from in vitro muscle strips, was increased 105% in MV animals co mpared to CON, but not different between CON and MV animals receiving Trolox. Lastly, th e chymotrypsin-like activity of the 20S proteaseome was elevated in the MV animals (+76%), but Trolox attenuated this rise in activity. These data indica te that Trolox supplementation during MV completely attenuates MV-induced contra ctile dysfunction and proteoly sis in the diaphragm.

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1 CHAPTER 1 INTRODUCTION Weaning patients from a mechanical vent ilator is a serious clinical issue. Mechanical ventilation (MV) is characteristica lly used to maintain alveolar ventilation in patients who are incapable of ventilation on their own. As such, MV is an important lifepreserving measure. However, removing patients from the ventilator, also known as weaning, can be difficult in many cases. Weaning procedures account for more than 40% of total MV time in patients who have diffi culty weaning from the ventilator (Esteban 1994), suggesting that this is a serious clinical issue. Dia phragmatic weakness, the result of atrophy, is a major cause of difficult wean ing. Therefore, the mechanisms underlying the rapid loss of diaphragm mass and strength during periods of MV should be explored. Background In many clinical situations, patients ar e unable to maintain adequate alveolar ventilation. In these cases, MV is necessary for life support. This may occur during acute respiratory failure, surgeries i nvolving general anesthesia, di seases such as sepsis, and with pre-term infants whose lungs and respir atory muscles are not completely developed. Unfortunately, removing a person from MV is not always simple since even short periods of MV can weaken the diaphragm to the poi nt where resumption of normal loading leads to diaphragm fatigue and resp iratory failure. The process of gradually weaning patients from a ventilator can result in extended hospi tal stays, as well as additional costs to the patients, insurance companies, and hospitals.

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2 The mechanism(s) behind MV-induced dia phragmatic weakness ar e unclear at this time. However, recent research by Powers a nd colleagues (2002) has reported that MVinduced diaphragmatic dysfunction is intrinsic to the muscle and increases in magnitude with increasing time on the ventilator. Oxidativ e stress is one potenti al mediator of MVinduced diaphragmatic dysfunction. Oxidativ e stress is the result of an imbalance between reactive oxygen species (ROS) produc tion and antioxidant protection (Lawler and Powers 1998), and has been implicated as a contributor in nu merous pathological conditions, including atheroscle rosis, obstructive lung diseas e, aging, and fatigue of skeletal muscle. Although ROS are continuously produced in human beings, a balance is generally maintained between ROS produc tion and cellular antioxidant systems. However, periods of stress, whether from tr auma, ischemia, infection, etc., lead to an increase in the formation of ROS, whic h may overwhelm antioxidant systems causing oxidative stress. This oxidative stress can cause lipid peroxidation, damage to DNA and proteins, and cell death. It is known that critical il lnesses like sepsis or adult respiratory distress syndrome can drastically increase the ROS production a nd lead to oxidative stress in skeletal muscle. This is significant because oxidized proteins are more prone to proteolytic attack and degradation (Grune et al. 1995, Grune et al. 1996, Dean et al. 1997, Nagasawa et al. 1997). A similar mechanism may contribute to protein loss in the diaphragm during MV. During periods of muscle disuse, ROS productio n has been shown to increase (Kondo et al. 1993a, Kondo et al. 1993b). Further, new data indicate significan t increases in lipid peroxidation and protei n oxidation in the diaphragms of mechanically ventilated rats (Shanely et al. 2002, Zergeroglu et al. 2003) and a corresponding increase in total in

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3 vitro protein degradation (Shane ly et al. 2002). Therefore, it seems logical that diaphragmatic weakening during MV-induced unloading may be caused by oxidative damage to contractile prot eins leading to heightened proteolytic degradation. Problem Statement Since prolonged controlled MV results in a significant loss of diaphragmatic maximal force production (LeBourdelles et al 1994, Powers et al. 2002, Radell et al. 2002, Sassoon et al. 2002, Yang et al. 2002, Capdev ila et al. 2003, Shanely et al. 2003), and is associated with evid ence of increased oxidative stre ss (Powers et al. 2002, Shanely et al. 2002, Zergeroglu et al. 2003), we postu late a causal relations hip and will seek to examine the efficacy of antioxidant infusion during MV for the preservation of diaphragm function. Trolox (6-hydroxy-2,5,7,8-te tramethylchroman-2-carboxylic acid), a water-soluble vitamin E analog, is an effective scavenger for a variety of radicals (Walker et al. 1998). This antioxidant prolongs surviv al of many cell types exposed to oxyradicals (Wu et al. 1991). Therefore, we will sp ecifically determine whether Trolox supplementation during 12 hours of controlled m echanical ventilation will attenuate MVinduced diaphragmatic contractile dysfunction, oxidative stress, and protein degradation. Variables in Study Independent variables. We will manipulate mechanical ventilation and Trolox supplementation. Dependent variables. We will measure diaphragm contractility, tyrosine release as a measure of degradation, 20S proteasom e activity, and markers of oxidative stress such as protein carbonyls.

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4 Control variable. We will only study female Sprague-Dawley rats, so gender is purposely excluded from this study. The animals will be young adult rats (~4 months old), thus maturation and aging effects are excluded from the study. Extraneous variable. We will not control PO2 levels in these animals, so hypoxic conditions will not be controlled. Pilot experi ments have been conducted to confirm that the MV protocol maintains normal arterial PO2 and PCO2 levels. However, the spontaneous breathing animals are expected to be mildly hypoxic and hypercapnic due to the effects of the anesthesia. To assess th e potential effects of these conditions on diaphragm function, a separate group of rats will be studied without exposure to MV or spontaneous breathing prot ocols (pure controls). Hypotheses We hypothesize that: 1.) Twelve hours of controlled MV will i nduce contractile dysfunction in the rat diaphragm compared to controls. 2.) Twelve hours of controlled MV will increase oxidative stress levels in the diaphragm muscle compared to control diaphragms. 3.) Twelve hours of controlled MV will increase the rate of total protein degradation within the diaphragm co mpared to control diaphragms. 4.) Infusion of Trolox during 12 hours of MV will attenuate the diaphragmatic dysfunction, reduce the rate of protein degrad ation, and decrease oxidative stress levels compared to controls. Definition of Terms Controlled mechanical ventilation (MV). Tracheostomized animals will receive all breaths from the volume-controlled smallanimal ventilator (Harvard Apparatus). The tidal volume will be ~1 ml/100 g body weight with a respiratory rate of 80 breaths/min.

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5 Positive end-expiratory pressure of 1 cm H2O will be used throughout the protocol. Therefore, the diaphragm muscle will be effectively unloaded. Spontaneous breathers (SB). Animals receiving sham surgeries and 12 h of anesthesia, without controlled MV. Antioxidant. A compound capable of preven ting or delaying damage from oxidative stress. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). a watersoluble vitamin E analog with antioxidant characteristics. Reactive oxygen species (ROS). Molecules derived from molecular oxygen that have an unpaired electron in their outer orbital, making them highly reactive. Oxidative stress. An imbalance between a greater production of reactive oxygen species and reduced antioxidant protection. Proteolysis. The process whereby proteins are broken down into peptide fragments and amino acids. This occurs th rough three main pathways: (1) lysosomal proteases (cathepsins), (2) Ca2+-dependent cysteine proteases (calpains), and (3) the ATPdependent, ubiquitin-proteasome pathway. Ubiquitin. A protein found in all cell types th at acts as a molecular tag when attached to proteins, marking them for degradation by the proteasome. Tyrosine. An amino acid that is neither synt hesized nor degraded in skeletal muscle. The net accumulation of this amino acid, assayed fluorometrically within the incubation buffer, reflects net in vitro protein degradation within muscle.

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6 Limitations/Delimitations/Assumptions Limitations. The invasive nature of this re search negates the use of human subjects. A rat model has been chosen to study the diaphragm muscle because of the similarities in structure a nd function between the rat dia phragm and human diaphragm. Trolox does not readily dissolve in salin e. Addition of sodi um hydroxide (NaOH) was necessary to solubilze Trolox. This in creased the pH of the Trolox solution well above the physiological range. As a result, the spontaneous breat hing group receiving Trolox was limited to only 3 animals that surv ived the 12-h protocol. These animals were not included in statistical analyses, thus we were limited in our ability to control for Trolox infusion without MV. Delimitations. Gender and species differences may exist in regard to the efficacy of Trolox as a protectant against diaphrag matic dysfunction. We have chosen to study only female Sprague-Dawley rats. Assumptions. It is assumed that the diaphragm is completely unloaded during MV. Previous experiments have inserted electromyographic (EMG ) needles into the muscle and found that it is silent during controlled MV (Le Bourdelles et al. 1994, Powers et al. 2002). Significance of the Study Atrophy and protein degradation occurring within an unloaded diaphragm muscle result in an 18% reduction in force generati on with just 12 hours of MV (Powers et al. 2002). This force loss increases to 46% with 24 hours of MV. Eighteen hours of MV is associated with a significant increase in lip id peroxidation and prot ein oxidation (Shanely et al. 2002). This research will improve our knowledge of the mechanisms associated with MV-induced diaphragmatic dysfunction. It will provide insight into clinical

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7 strategies using antioxidants to attenuate diaphragmatic at rophy incurred during MV so that patients may be removed more swiftly and successfully from the ventilator.

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8 CHAPTER 2 LITERATURE REVIEW The diaphragm is an essential muscle for the maintenance of normal ventilation in mammals. This muscle has an activity level greater than most other skeletal muscles, which puts it at increased risk for atrophy and dysfunction during prolonged periods of inactivity such as mechanical ventilation (MV) The purpose of this study is to determine whether Trolox supplementation during MV will attenuate MV-induced diaphragmatic dysfunction related to oxidativ e stress and protein degrada tion. This chapter provides a critical review of the scientif ic literature related to the proposed project. All pertinent articles in the specified areas will be covered. In some cases, however, the literature abounds and only a few representa tive articles will be reviewed in detail with cursory reference to other corroborating evidence. Wh enever possible, interpretations of the reviewed data will be offered based on perceived consensus in the literature. This review is organized under the foll owing headings: (a) Skeletal muscle adaptations to unloading, (b) Oxidative stress and skeletal muscle, (c) Skeletal muscle unloading and protein degrada tion, and (d) Antioxidant supplementation and skeletal muscle. Skeletal Muscle Adaptations to Unloading Muscle atrophy, the result of disuse, deve lops rapidly after immobilization (Appell et al. 1997). Several models of disuse exist that either prevent th e loading of skeletal muscle with normal body weight, or eliminat e the effect of gravity on the upright position. These models include hindlimb unloadi ng in rats, casting of limbs in animals

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9 and humans, spaceflight or simulation of a microgravity environment, bed rest, and denervation. Atrophy and Fiber Type Shifts with Unloading Fast and slow twitch locomotor muscles undergo considerable atrophy with unloading (McDonald and Fitts 1995). Simultaneously, unloaded locomotor muscles exhibit a fiber type conversi on from type I to type II fi bers (Haida et al. 1989). The greatest change occurs in antig ravity, slow twitch muscles such as the soleus. After 1, 2, and 3 wk of hindlimb unloading (HU) in Spra gue-Dawley rats, mean mass of the soleus was decreased by 28, 44, and 56%, respectively (McDonald and Fitts 1995). Mean fiber diameter decreased with incr easing length of HU. Riley et al. (1990) demonstrated that 14 days of unloading caused a reduction in type s I and IIa cross-sec tional areas (CSA) of 63 and 47%, respectively. They also showed that HU reduced the muscle-to-body weight ratio showing muscle-specific eff ects of the unloading treatment. A decrease in the percentage of slow twitch fibers, with an increase in the percentage of fast twitch fi bers, leads to an increase in maximal shortening velocity (Canon and Goubel 1995). A 4 wk hindlimb susp ension study led to significant fiber atrophy in both the soleus and extensor digi torium longus (EDL) muscles (Deschenes et al. 2001). A significant decrease in the percentage of type I fibers was noted in unloaded solei. This was accompanied by an increase in the percentages of types IIa and IIx/b fibers. The loss of fiber size is of concern be cause force production is directly related to fiber size. Muscle unloading failed to induce significant fibe r type conversi on within the fast twitch EDL muscle. Like hindlimb unloading, casting, or lim b fixation, results in muscle atrophy (Booth 1977, Kondo et al. 1993a, Kondo et al. 19 93b, Appell et al. 1997). Both the onset

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10 and the degree of atrophy of limb muscles during casting immobilization are dependent on the length of the muscle during limb fi xation (Booth 1977). Booth (1977) performed two sets of experiments using a rat model, one with the ankl e and foot fixed in slight plantar flexion (PF), and one with the foot fixed in dorsal flexion (DF). In PF, the calf muscles are slightly less than resting length, while they ar e slightly lengthened in DF. Weight losses of the gastrocnemius, soleus quadriceps, and white portion of the vastus lateralis were exponential between days 2-10 of PF immobilization. During 28 days of PF immobilization, the gastrocnemius muscle at rophied by 51%, and the plantaris atrophied by 48%. In contrast, there was no change in the weight of the stretche d tibialis anterior muscle during PF immobilization. When the ankle was casted in DF, the calf muscles were stretched beyond resting length and a delay in the onset of atrophy was noted. Appell et al. (1997) reported si milar findings. Eight days of casting in male Wistar rats with the soleus muscle in a shortened positi on lead to a 35% atrophy compared to control muscles. These data clearly demonstrate that a greater degree of atrophy, as well as an earlier onset of atrophy, is seen in muscles fi xed in positions that are less than resting length. Seven days of casting the ankle joint of one hindlimb in the fully extended position resulted in a loss of ~45% of soleus musc le weight (Kondo et al. 1993a). In a similar experiment, 4-, 8-, and 12-day immobili zation of one hindlimb by casting led to decreases of 49, 60, and 81% of control soleus muscle weight, respectively (Kondo et al. 1993b). The activity of xanthine oxidase (XOD) increased significantly in the atrophied muscles. Type O (superoxide-producing) XOD was ~2.3 times higher in immobilized muscle than in control muscle. Also, the s ubstrates of XOD, xa nthine and hypoxanthine,

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11 increased in atrophy. Therefore, superoxide-g enerating XOD may be more active in the atrophied muscle, meaning oxidative stress is accompanying atrophy. These observations formed the basis of our original hypothesis th at oxidative stress may be associated with diaphragmatic atrophy. Adaptations in the size, metabolic properties, and vascularity of muscle fibers occur in space where the gravity-dependent load of the body on muscles is absent (Edgerton et al. 1995). Vastus lateralis muscle fibers sampled from astronauts before and after spaceflights showed postflight biopsies with 6-8% fewer type I fibers than preflight. This loss of type I fibers seemed to be accounted for by an increase in type IIa fibers. Mean fiber CSAs were 16-36% smaller after the fli ght. Little difference in percent atrophy was found in type I versus type II fibers. The number of capillaries per fiber was 24% lower after flight as compared to before flight Spaceflight resulted in an increase in the myofibrillar ATPase activity of type II fibers, whereas alpha-glycerophosphate dehydrogenase (GPD) activity was 80% higher in type I fibers after flight. This study found significant variability between subjects th at was attributed to the volume and kind of physical work that each astronaut performed during flight. The results indicate that the degree of atrophy may have been related to the type of physical activities undertaken during spaceflight. Contractile Dysfunction with Unloading Maximal isometric specific tension (Po) of the soleus was significantly reduced after 1 and 2 wk of hindlimb suspension comp ared to normal loaded solei in SpragueDawley rats (McDonald and Fitts 1995). A re duction in the number of cross bridges per fiber area, and possibly a reduced force per cros s bridge, may explain th e decrease in Po.

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12 The gravity-dependent load of the huma n body in the upright position is essential for maintenance of lower limb skeletal muscle function (Berg et al. 1997). In particular, contractile properties of sl ow, antigravity skeletal muscle are sensitive to the microgravity environment. Six days of spacef light induced contractile changes in the soleus muscles of male Sprague-Dawley rats (Caiozzo et al. 1994) The force-velocity relationship, force-freque ncy relationship, and fatigability were studied in situ 3 h after landing. Maximal isometric tension (Po) wa s decreased by 24% and maximal shortening velocity was increased by 14% in flight mu scles. The flight muscles’s force-frequency curve was shifted to the right of the control muscles’s curv e. Control muscles generated 64% of the initial Po after the fatigue protoc ol, while flight muscles only generated 36% of initial Po. Bed rest results in similar changes in sk eletal muscle function. After 6 wk of bed rest in 7 healthy men, maximu m voluntary isometric and concen tric knee extensor torque decreased across angular velo cities by 25-30% (Berg et al 1997). Type I fiber crosssectional area (CSA) of the vastus latera lis decreased by 18.2%. No change in CSA or fiber diameter was apparent in either type IIa or IIb fibe rs. The greater loss in strength compared to muscle CSA suggests specific te nsion of muscle and/ or neural input to muscle is reduced. Another study showed that 20 days of bed rest decreased maximal knee extension force by 10.9% (Kawakami et al 2001). The reduction of muscle strength in this study was likely due to a decr eased ability to activate motor units. In conclusion, antigravity, slow twitch muscles such as the soleus are more susceptible to changes as a result of unloa ding than are nonpostural, phasic muscles such as the EDL. These changes include fibe r atrophy, reduced force production, and a

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13 conversion to a faster type muscle. A decr eased number of active cross bridges per volume of muscle after unloading may explain the loss in specific tension seen in humans and animals. Mechanical Ventilation and Diaphragm Atrophy An early epidemiological study found that some infants and neonates who had received long-term ventilatory assistan ce (>12 days) had subnormal diaphragmatic muscle mass on gross necropsy examination (K nisely et al. 1988). The retrospective study examined sections of the costal diap hragm, along with portions of the infrahyoid strap muscle and the posterior portion of the tongue. These extra-diaphragmatic sites appear to be coordinated with diaphragmatic function in infants. Hi stologic findings in the diaphragms of neonates and infants s upported by MV for at least 12 days were consistent with disuse atrophy, denervation atrophy, or failure of normal growth and maturation. Myofibers from the other two s ites appeared normal The researchers concluded that long-term ventilatory assist ance predisposes diaphragmatic myofibers to disuse atrophy or to failure of normal growth. This weakening may play a role in difficult weaning procedures from ventilatory support. Thus, mechanical ventilation unloads the diaphragm muscle like hindlimb suspensi on, casting, spaceflight, bed rest, and denervation unload other ma jor skeletal muscles. Several studies have shown that controll ed MV leads to diaphragmatic atrophy (Le Bourdelles et al. 1994, Shanely et al. 2002, Ya ng et al. 2002, Capdevila et al. 2003) and reductions in protein content (Shanely et al. 2002). Shanely et al. (2002) observed a significant decrease in both total and costal diaphragm masses after 18 h of MV in rats, but no losses of body mass or so leus mass. All four diaphr agmatic myosin heavy chain (MHC) types experienced a reduc tion in CSA. However, type IIx and IIb fibers atrophied

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14 to a greater extent than type I fibers. This c ontrasts with results seen in locomotor muscle during periods of atrophy, where type I fibers typically atrophy more than type II fibers. In rats mechanically ventilated for up to 4 days, a significant de crease in diaphragm weight / body weight was seen, which amounted to a mean reduction of 13.4% compared to controls (Yang et al. 2002). These resear chers noted a shift in myosin heavy chain (MHC) isoform from slow-to-fast. There was a decrease in the percentage of fibers expressing type I MHC, while the number of fibers co-expressing both type I and type II MHC increased in the diaphragm (12.5% vs. 3% in controls). In cont rast, the percentages of type I, type II, and hybrid fibers re mained unchanged in the limb muscles after MV. The combination of mechanical unloading, re duced electrical activ ity, and intermittent passive shortening in the diaphragm duri ng MV may be powerful stimuli for MHC transformations. These modifications may a lter the maximal specific force and fatigue resistance of the diaphragm following MV. In contrast, Sassoon et al. (2002) found th at 3 d of MV did not alter fiber type proportions or their relative c ontribution to total CSA in a ra bbit model. Like Sassoon et al. (2002), Capdevila and colleagues (2003) found no significant alterations in fiber type proportions following 513 h in the rabbit di aphragm. It is possible that species differences contributed to th e discrepancy between these st udies and Yang et al. (2002) since fiber type composition differs in rat and rabbit diaphragms. Mechanical Ventilation-induced Contractile Dysfunction Eight studies have been published whic h confirm that controlled mechanical ventilation alters diaphragm contractile properties. Alt hough different animal models were employed, all of these studies agree th at MV significantly reduces diaphragmatic force-generating capacity. One of the earliest studies, performed by Le Bourdelles et al.

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15 (1994), found that 48 h of MV in rats significantly decreased in vitro diaphragm contractility compared to controls, while the soleus and EDL muscles’s contractility were unaffected. No electrical activity of the di aphragm was detected during MV in the 2 animals in which it was measured. Diaphrag matic force-generating capacity was reduced by 41.5% compared to spontaneously breathi ng controls. The data did not show a difference in total protein cont ent, or citrate synthase or lactate dehydrogenase enzyme activities between control and MV diaphragms. Their data indicate that the decreased force generation did not result from decreased muscle mass as typically seen with general disuse atrophy. An important finding from this study was that the level of sodium pentobarbital required to maintain a surgical plane of anesthesia over a 2-day period did not induce locomotor muscle atrophy, nor di d it impair locomotor muscle maximal tetanic force generation. This is significant b ecause it demonstrates that MV itself exerts deleterious effects on diaphragmatic f unction independently of anesthesia. Anzueto et al. (1997) mechanically ventil ated adult baboons (n=7) for 11 days and showed that maximum transdiaphragmatic pre ssure decreased by 25% from day 0 to day 11. In addition, diaphragmatic endurance decreased by 36% from day 0 to day 11. These animals were infused with a long-acting neuromuscular blockade, pancuronium (10 g/kg/h), which may have contributed to the results. However, pancuronium was withheld on day 11, and reversal of neur omuscular blockade was noted. No major changes occurred in hemodynamics, oxygenation, or lung function. Arterial pressure, pulmonary artery pressure, pulmonary arte ry occlusion pressure, and cardiac output remained constant over the 11-day period. The absolute force-frequency curves showed a

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16 decrease in diaphragmatic response to all frequencies of stimulation tested. Prolonged MV in this baboon model resulted in impair ed diaphragmatic strength and endurance. Five days of volume-controlled MV in a piglet model resulted in depressed diaphragm contractility and activation (Rad ell et al. 2002). However, nerve conduction and transmission were unaffected. Bipolar tr ansvenous pacing catheters were used to stimulate the phrenic nerve and pace diaphr agm contractions during measurements. The researchers found that transdiaphragmatic pr essure (Pdi) decreased over time at all frequencies tested. By day 5, the drop in Pd i was greater than 20% at all frequencies. There was a 30% decrease in compound muscle action potential (CMAP) amplitude of the costal diaphragm from day 1 to day 5. Th e stable response to repetitive stimulation does not support neuromuscular transmission fa ilure as the cause of dysfunction. Instead, the decrease in CMAP amplitude and fall in force output are indicative of excitationcontraction (E-C) coupling or membrane de polarization as mechanisms leading to diaphragmatic dysfunction. Thus, this study found that nerve conduction and neuromuscular transmission are unaffected during prolonged MV but the diaphragm does experience a loss in function that may originate at the level of the muscle cell membrane or the contractile apparatus. Yang et al. (2002) also found that maxi mal twitch and tetanic force generating capacity were significantly lowered in rats m echanically ventilated for up to 4 days as compared to controls. There were no signifi cant differences in c ontraction time, halfrelaxation time, or fatigue resi stance. Optimal muscle length, Lo, was found to be shorter in the MV group compared to controls and anesthetized spontaneous breathers. This may provide indirect evidence for a loss of sarcomeres in series.

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17 A study by Powers et al. (2002) examined the time course of MV-induced diaphragmatic contractile dysfunction in an in vitro diaphragm strip preparation. When compared to control rats, MV of 12, 18, and 24 h duration resulted in a right shift in the force-frequency curve of the diaphragm. Th e magnitude of the curve’s right shift was dependent upon the duration of MV. Twelve h of MV resulted in an 18% reduction in the mean diaphragmatic specific tension, while 24 h of MV resulted in a 46% reduction. This experiment also included two groups of spontan eously breathing (SB) animals that were maintained on a surgical plane of anesthesia for 18 and 24 h. Analysis of arterial blood gas tensions and pH revealed that these groups experience d hypoxemia, hypercapnia, and mild acidosis. These disturbances were likely due to hypoventilation resulting from depressed ventilatory drive. Ne vertheless, like Le Bourdelles and coworkers (1994), this lab found that the level of pentobarbital sodium required to maintain a surgical plane of anesthesia did not impair in vitro diaphragmatic function in the spontaneously breathing animals. The researchers concluded that MV -induced contractile dysfunction was due to intrinsic changes within diaphragm fibe rs. These may include a reduction in the myofibrillar protein concentra tion, abnormalities of contractile or cytoskeletal proteins, and/or impaired excitationcontraction (E-C) coupling. Controlled MV also had a time-depende nt deleterious effect on diaphragm contractility in a rabbit model (Sassoon et al. 2002). Transdiaphragmatic pressure decreased to 63% of controls after 1 day of MV, and to 49% after 3 days. Similarly, in vitro tetanic force decreased to 86% of control values after 1 day, and to just 44% of controls after 3 days. A major finding of this study was that diaphragm muscle injury accounted for 66% of the variance in the reduction of tetanic force. Significant myofibril

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18 damage was found in diaphragms after 3 days of MV, but not in soleus muscles from the same animals. There was no myofibril damage in control diaphragms. Another study in rabbits mechanically ventilat ed for 49 1 h also found ev idence of altered diaphragm fiber ultrastructure indicative of fiber injury in the MV group (Berna rd 2003). Disruption and fragmentation of myofibrils were obser ved in diaphragms after MV, along with an increase in the size of the interfibrillar sp ace and in the size and number of sarcoplasmic lipid vacuoles. The mechanism for myofibril injury with inactivity is unknown, but may contribute to MV-induced diaphragmatic dysf unction and difficulties in weaning patients from ventilators. The laboratory of Capdevila et al. (2003) examined both the diaphragm and 5th external intercostal muscle following 513 h of MV in rabbits. MV significantly decreased Po compared to controls by 25% This was significantly worsened after a fatigue run. Diaphragmatic and 5th external in tercostal muscle masses were significantly reduced following MV. The MV rabbits had lo wer peak tetanic tensions, reduced fatigue resistance indices, and increas ed relaxation times compared to control diaphragms. The force reduction of the diaphragm was most likely related to the change in mass. Eighteen h of controlled MV in a rat model significantly reduced both diaphragmatic maximal twitch force producti on and Po (~20%) compared to controls (Shanely et al. 2003). However, diaphragms from MV-treated animals maintained a significantly greater fatigue resistance compared to control animals. That is, MV diaphragms maintained higher relative for ces throughout a 30-min fatigue test. When absolute force production was compared, how ever, the control diaphragms produced higher specific forces than MV diaphragms during the fatigue test. Interestingly, costal

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19 diaphragm citrate synthase, total superoxi de dismutase (SOD), Cu-Zn-SOD, and MnSOD activities were significantly greater in the MV animals than the control animals. These findings indicate that 18 h of MV improves diaphragmatic fatigue resistance relative to maximal force. However, long duration MV (weeks to months) does impair diaphragmatic endurance. Oxidative Stress and Skeletal Muscle Reactive oxygen species (ROS ) production results from a number of biochemical reactions, most notably aerobic metabo lism (Lawler and Powers 1998). Infection, inflammation, strenuous exercise, and obstructiv e lung disease are a few conditions that increase diaphragm exposure to ROS. Skeletal Muscle Atrophy and Dysfunction Related to Oxidative Stress Kondo et al. (1993a) have found that musc le atrophy induced by immobilization is accompanied by oxidative stress. Thiobarbitu ric acid reactive substance (TBARS) and oxidized glutathione (GSSG) we re increased, while total gl utathione (GSH) was reduced in atrophied soleus muscle from immobili zed hindlimbs. The ankle of one hindlimb of male Wistar rats was immobilized with the soleus muscle in a shortened position. Some rats were sacrificed afte r 7 days of immobilization (A trophy group), while the ankle joints of other rats were remobilized for another 5 days (Recove ry group). The TBARS level in atrophic muscle increased significantl y in the Recovery group, indicating a rise in lipid peroxidation. GSSG levels were significantly greater in atrophic muscle than that in contralateral control muscle. Total GSH leve l decreased significantly in atrophic muscle. Both the increase of TBARS and GSSG impl y enhanced oxidative stress during muscle recovery from atrophy.

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20 The diaphragm muscle is susceptible to alte rations in contractility resulting from a direct effect of hydroxyl radical (O H) and superoxide anion radical (O2 •) on contractile proteins (Callaha n et al. 2001). Chemically sk inned (Triton X-100) single rat diaphragm fibers exposed to O2 • had a significant 14.5% reduction in maximum calcium-activated force. Exposure to OH significantly decreased maximum calciumactivated force by 43.9%. Hydrogen peroxide (H2O2) did not affect maximum force or calcium sensitivity. The effects of OH and O2 • on contractility may contribute to the characteristic respiratory muscle dysfunction seen in certain pathophysiological conditions such as sepsis and skeletal muscle fatigue. When proteins are damaged by ROS, their function is impaired, and their susceptibility to proteolysis is enhanced (Nagasawa et al 1997). Oxidatively modified proteins are easily degraded by the proteasom e, a multisubunit proteinase. Rats given an intraperitoneal injecti on of ferric nitrilotria cetate (FeNTA) and sacr ificed at 1.5, 3, and 6 h after injection had si gnificant modification of muscle proteins after an iron overload (Nagasawa et al. 1997). Prot ein carbonyl content of both soleus and EDL muscles was elevated up to 3 h after injection. These results show that muscle pr oteins were modified by free radicals generated from the FeNTA inj ection. The rate of tyro sine release reached a maximum at 3 h after injection, suggesting an increase in the ra te of total protein degradation up to 3 h after th e onset of an oxidative stre ss. Myosin and actin responded strongly to specific antibody against 2,4-dinitrophenyl gr oup, implying that these myofibrillar proteins were dras tically modified by free radicals. These results suggest that oxidatively modified muscle prot eins undergo rapid proteolysis.

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21 MV-induced Oxidative Stress Shanely et al. (2002) found that oxida tive stress, specifically protein carbonyl content and total 8-isoprostane concentration, was increased after 18 h of MV in rats. Protein carbonyl levels signifi cantly increased by 44%, while 8-isoprostane concentration increased 53% compared with controls. Shor t-term controlled MV of just 6 h was sufficient to increase oxidative injury in the diaphragm of ra ts (Zergeroglu et al. 2003). Reactive protein carbonyl deri vatives (RCD) and lipid hydroperoxides were increased after 6 and 18 h of MV. RCD accumulation wa s limited to insoluble proteins with molecular masses of ~200, 120, 80, and 40 kDa. Ox idative stress is therefore evident in the diaphragm following MV, and is a potenti al mediator of MV-induced diaphragmatic dysfunction by way of increas ed protein degradation. Skeletal Muscle Unloadin g and Protein Degradation In skeletal muscle, the balance between protein synthesis and protein degradation determines whether muscle growth or at rophy will occur (Baracos et al. 1986a, 1986b). Protein degradation, specifically myofibrill ar protein degradation, may lead to the dysfunction seen in the diaphragm after MV Three major pathways exist for general protein degradation: lysosomal proteases, calc ium-activated calpains, and the proteasome complex. Many studies indicate that the prot easome is responsible for ~70-80% of the increased cellular protein degradation follo wing an oxidative stress (Grune et al. 1995,1996, Grune and Davies 1997). More specifi cally, it appears the 20S proteasome is responsible for the degradation of oxidized proteins since the 26S proteasome is inhibited/inactivated by oxidative stress (Rei nheckel et al. 1998). Recognition of exposed hydrophobic patches is the proposed mechanis m by which the proteasome selectively degrades oxidatively modified proteins (Grune et al. 1997).Oxidative damage to a protein

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22 leads to partial unfolding and exposure of normally shielded inte rnal hydrophobic patches that are recognized by the proteasome, which cat alyzes the degradation of that protein. Net protein degradation can be estimated from the rate of release of free tyrosine from tissue proteins (Lowell et al. 1986). Tyrosine is neither synthesized nor degraded in skeletal muscle, so the net accumulation of th is amino acid is directly related to net degradation of cell protein. Ty rosine and 3-methyl histidin e (3-MH) release have been used to assess total protein degradati on and myofibrillar protein degradation, respectively, under a variety of treatments. For example, Lo well et al. (1986) used both techniques to identify a differential br eakdown of myofibrillar and nonmyofibrillar proteins during starvation. Like wise, using a model of denerv ation atrophy, Furuno et al. (1990) have shown that overall protein breakdown is greater in denervated solei than in contralateral controls. Treatments that block the lysosomal and Ca2+-dependent proteolytic pathways did not attenuate protein breakdown, suggesting proteasomedependent proteolysis may account for dene rvation-induced loss of protein. Lastly, muscle length appears important in determin ing rates of protein de gradation (Baracos et al. 1986a). Muscles fixed at resting length (Lo) in situ experienced the lowest rate of protein breakdown compared to unrestrained muscles. The unrestrained solei and EDLs shortened spontaneously and had 25-45% grea ter net protein degradation than muscles fixed at Lo. Nine days of hindlimb suspension lead to atrophy (-55%), loss of protein (-53%), and elevated protein breakdown (+66%) in ra t soleus muscles compared to controls (Taillandier et al. 1996 ). A non-lysosomal, Ca2+-independent proteolytic pathway accounted for the increased proteolysis a nd muscle atrophy. This study suggests that

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23 ATP-ubiquitin-dependent proteolysis due to th e proteasomal pathway is responsible for the majority of the increased protein degr adation and muscle atrophy in unweighted hindlimb muscle. Eighteen h of MV resulted in significant reductions in diaphragmatic protein content, and significant increases in total in vitro protein degradation, as measured by the rate of tyrosine release from diaphragm st rips (Shanely et al. 2002). The rate of diaphragmatic protein degradation was increas ed by 28% after MV compared to controls. The significant increase in diaphragmatic proteolysis following MV could be reduced following the addition of either a proteasome i nhibitor (lactacystin) or an inhibitor of both calpain and lysosomal proteases (E64d). Th ese researchers also found that oxidative stress was elevated in the diaphragm followi ng 18 h of MV. Therefore, MV results in an increase in oxidative stress (Shanely et al. 2002) that leads to the oxidation of proteins, making them more susceptible to proteolytic attack and degradati on (Grune et al. 1995, Grune et al. 1996, Naga sawa et al. 1997). Antioxidant Supplementation and Skeletal Muscle Muscle cells contain complex defense m echanisms to protect against oxidative stress (Powers and Hamilton 1999). The two classes of endogenous protective mechanisms are: 1) enzymatic and 2) nonenz ymatic antioxidants. Important enzymatic antioxidants include superoxi de dismutase (SOD), glutathi one peroxidase (GPx), and catalase (CAT). These are responsible fo r removing superoxide radicals, hydrogen peroxide or organic hydrope roxides, and hydrogen peroxi de, respectively. Important nonenzymatic antioxidants include vitamins E and C, beta-carotene, glutathione (GSH), and ubiquinone.

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24 In vitro experiments using excise d animal muscle have s hown that addition of antioxidants can delay fatigue and improve muscular performance (Shindoh et al. 1990, Reid et al. 1992). For example, N-acetylcyst eine (NAC) has been shown to protect an in situ rabbit diaphragm strip preparation from oxi dative injury during periods of rhythmic repetitive isometric contraction (Shindoh et al. 1990). The research ers postulate that NAC, a potent radical scavenger, may have a ffected fatigue by preventing free radicalmediated damage in the exercising diaphrag m muscle. Similarly, Reid et al. (1992) found improved muscular performance in rat diap hragm fiber bundles with both SOD and CAT supplementation. These antioxidants inhibited low-frequency fatigue but did not alter high-frequency fatigue. The effects of antioxidant supplemen tation on human performance are less definitive. Many studies using human subjects have experimental design weaknesses, and most have only investigated th e effects of a single antioxida nt rather that combining both lipid-soluble and water-sol uble antioxidants (Powers a nd Hamilton 1999). Few studies show improved human exercise performance with antioxidant supplementation. However, the laboratory of Reid and colleagues (1994) has shown that NAC administration in human subjects improves muscular endurance during low-frequency electric stimulation. During fatiguing cont ractions at 10 Hz, NAC increased force production by ~15%. However, NAC had no effect on fatigue induced by 40 Hz stimulation, or on recovery from fatigue. Additional research is required to determine the specific effects of supplemental antioxidant s on humans. Careful research design and understanding of bioavaila bility are essential to draw conclusions.

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25 Vitamin E The most widely studied antioxidant is vitamin E, or alpha-tocopherol (Powers and Hamilton 1999). Kondo et al. (1993a) injected either vitamin E or placebo one time daily into Wistar rats with one hindlimb immobilized for 7 days with the soleus muscle in a shortened position. The TBARS level of atr ophic muscle in the vitamin E group was significantly less than in the placebo group. The muscle weight was significantly greater, and the degree of atrophy was significantly reduced by ~20% in the vitamin E group compared to the placebo group. Intraperitoneal injections of vitamin E during periods of muscle atrophy effectively served as an anti oxidant to reduce oxidati ve stress and prevent muscle atrophy. Eight days of immobilization led to a 35% atrophy in the hindlimb of rats (Appell et al. 1997). However, when vitamin E was supplemented, the muscles atrophied by only 12%. Control muscles of those animals supplem ented with vitamin E contained even less of the oxidized form of glutathione (GSSG) th an baseline oxidative stress. These results indicate that the soleus musc le atrophies to a le sser extent when supplemental vitamin E is given during a period of disuse. Trolox Vitamin E is a natural antioxidant, but is extremely lipophilic and is taken up slowly by cells (Zeng et al. 1991) It is therefore not an adeq uate therapeutic antioxidant. Trolox (6-hydroxy-2,5,7,8-tetramethylchroma n-2-carboxylic acid), is a hydrophilic vitamin E analog synthesized in 1974 by Sco tt and colleagues (1974). Trolox differs from vitamin E by the absence of the phytyl side chain, which makes Trolox water-soluble (Klein et al. 1991).

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26 Trolox is an effective scavenger of radical s (Walker et al. 1998). This antioxidant has been shown to prolong the survival of cells exposed to oxyradicals (Wu et al. 1990, Wu et al. 1991). Specifically, it was found that Trolox protected human ventricular myocytes and hepatocytes against oxyradicals genera ted by xanthine oxidasehypoxanthine and prevented lysis of erythrocytes exposed to an azo-inhibitor (Wu et al. 1990). The protection by Trolox was dose depend ent in all cell types, and surpassed the antioxidant capabilities of ascorbic aci d, SOD, and CAT. Using hepatocytes, the researchers determined that Trolox behaved mech anistically as an antioxidant in cells. In cultured rat hepatocytes, 0.5-16 mmol/L Trolox prolonged the survival of cells exposed to xanthine oxidase-hypoxanthine oxy radicals (Wu et al. 1991). Optimum levels of Trolox were between 1 and 2 mmol/L. Pr otection by Trolox surpassed that provided by ascorbate, mannitol, SOD, and CAT. This la boratory also studied a global and partial model of hepatic ischemia-reperfusion in ra ts (Wu et al. 1991). Infusion of Trolox (7.5-10 mol/kg body weight) prior to reflow reduced liver necrosis by more than 80% compared to control, untreated animals. These data indicate a strong and rapid antioxidant-like action by Trolox on rat hepatocytes a nd postischemic-reperfused rat liver. Trolox also protected regionally ischemic, reperfused porcine hearts against free radical generation (Klein et al. 1991). Speci fically, Trolox reduced free radical generation from stimulated neutrophils by 30% in the treatment group before ischemia and immediately before reperfusion. After 3 days of reperfusion, recovery of regional function had improved to a significantly greater extent in the treated group than in control hearts. Mean recovery of systolic shorteni ng amounted to 10% of the baseline value in

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27 the control animals, and to 28% in the Tr olox group. Trolox did not reduce infarct size, but did accelerate functional re covery in ischemic, reperf used porcine hearts. Trolox has also been shown to improve the long-term storage of isolated skeletal muscle (van der Heijden et al. 2000). Si gnificant protection of contractile function occurred with addition of 1mM Trolox in th e bathing solution of soleus and cutaneus trunci muscles from the rat. These muscles were stored for 16 h at 4oC. Trolox effectively reduced the overproducti on of oxyradicals. Trolox treatment in vivo protected methylmercury (M eHg)–treated rat skeletal muscle from many of the clinical manifest ations of MeHg-intoxi cation (Usuki et al. 2001). Trolox prevented decreases in mitochondr ial enzyme activities in soleus muscle, repressed apoptosis in cerebe llum, and protected against th e decrease in glutathione peroxidase activity of the soleus following MeHg-intoxication. Summary The removal of weight bearing from skelet al muscle leads to rapid and significant atrophy. The rat model of mechanical vent ilation effectively un loads the diaphragm muscle, thereby causing atrophy. Oxidative st ress is evident in unloaded muscle, including diaphragm muscle fr om mechanically ventilated animals, and may increase rates of protein degradation. Interventions with antioxidants have shown that muscle atrophy and dysfunction can be attenuated during unloading. The antioxidant properties of Trolox make it an appealing subject for research. No studies have given Trolox to animals to prevent muscle dysfunction duri ng unloading. This project will determine whether mechanical ventilation-induced diap hragmatic dysfunction, ox idative stress, and proteolysis can be attenuat ed with supplementation of the antioxidant-like compound, Trolox.

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28 CHAPTER 3 METHODS Experimental Design The following groups were formed to complete these experiments: Pure control group CON n=8 Spontaneous breathers SBS n=8 Mechanical ventilation MVS n=8 Spontaneous breathers receiving Trolox SBT n=3 Mechanical ventilation receiving Trolox MVT n=8 Animals The subjects were adult (~4 month-old) female Sprague-Dawley rats (~250 g). All were housed in the J. Hillis Miller Animal Sc ience Center and fed th e same diet (rat chow and water ad libitum ) for one week prior to the experi ment. Animals were maintained on a 12 h light:dark photoperiod. All proce dures followed NIH guidelines and were approved by the University of Florid a’s Animal Care and Use Committee. General Procedures After a period of acclimation (1 week), ra ts were randomly assigned to one of the 5 groups listed above. The control group (CON) animals were free of intervention before removal of the diaphragm for measuremen ts. These animals received an acute intraperitoneal inject ion of sodium pentobarbital ( 65 mg/kg body weight). When a surgical plane of anesthesia was reached, the diaphragms were removed for measurement of in vitro contractile properties and tyrosine rele ase, and remaining muscle was frozen for biochemical assays.

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29 Animals in the 2 mechanically ventilated groups (MVS and MVT) were given an intramuscular injection of gl ycopyrollate (0.04 mg/kg) to re duce respiratory secretions during the protocol. Thirty min later, animals were anesthetized with an intraperitoneal injection of sodium pentoba rbital (65 mg/kg). Upon reac hing a surgical plane of anesthesia (no ocular response, no hind limb withdrawal response), they were tracheostomized by an experienced lab technician and mechanically ventilated with a volume-cycled ventilator (Harvard Apparatu s). The tidal volume was established at ~1 ml/100 g body weight with a respiratory rate of 80 breaths/min. Positive end-expiratory pressure of 1 cm H2O was used for all MV animals. Throughout the MV period, heart activity, blood pressure, and co re temperature were monitored. A lead II ECG displayed electrical activity of th e heart. A catheter placed in the carotid artery gave constant blood pressure readings. Core temperature was monitored with a rectal thermometer and adjustments were made to help anim als maintain body temperature at 37 + 1oC with a recirculating heating blanket. A catheter was placed in the jugular vein for the infusion of sodium pentobarbital (~10 mg/kg/h) and Trolox. In the MVT group, th e jugular vein was cannulated before the carotid artery and before the tracheotomy. A priming dose of Trolox (20 mg/kg) was infused over a 5 min period. Twenty min later, MV was started, along with the constant infusion of Trolox at a rate of 5 mg/kg/h. C onstant supervision was provided for the rats throughout the MV period. This included expressing the bladder, removing airway mucus, monitoring anesthesia rate, rotating the animals, and infusing saline to maintain hydration status. To reduce airway secretions glycopyrollate (0.04 mg/kg) was injected intramuscularly every 2 h.

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30 Spontaneously breathing (SBS and SBT) an imals were anesthetized in the same manner and received sham surgeries. Thes e animals were included in the study to determine whether long-term anesthesia (s odium pentobarbital) im pairs diaphragmatic contractile function. A tube was inserted into the trachea, and these animals were maintained on a surgical plane of anesthes ia for the 12 h period while continuing to breathe on their own. The carotid artery and jugular vein were cannulated, and sodium pentobarbital was infused for a 12 h peri od. However, these animals were not mechanically ventilated. The SBT group rece ived the same dose of Trolox as the MVT group (20 mg/kg priming dose, 5 mg/kg/h constant infusion). At the end of the 12 h experimental peri od, all rats were killed by injection of sodium pentobarbital (50 mg/kg) and the di aphragm was removed for immediate analyses of contractile function and protein degrad ation as described below. After obtaining muscle strips for contractile and protein de gradation measurements, the remaining costal diaphragm tissue was dissected, weighed, fro zen in liquid nitrogen, and stored at –80oC until needed. Diaphragm Contractile Function The entire diaphragm with the supporting ribs and central tendon was removed and placed in a dissecting chamber containing a Krebs-Hensleit solution aerated with 95% O2-5% CO2 gas. The entire crural diaphragm wa s removed and discarded. A strip was cut from the midcostal region including the cen tral tendon and the rib. This was secured vertically in an organ bath maintained at 24oC between two Plexiglass clamps. The muscle strip was placed between two platinum field electrodes connected to an isometric force transducer (model FT-03, Grass Instrume nts, Quincy, MA). This strip was mounted on a micrometer to allow for muscle length adjustment. A 15 min equilibration period in

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31 the bath preceded all data collection. Duri ng this time, the remaining diaphragm muscle was dissected and sectioned into 9 pieces: 2 dorsal, 6 midcostal, and 1 ventral section. All sections were blotted, weighed, frozen in liquid nitrogen, and stored at –80oC. All further analyses were conducted on midc ostal diaphragm sections. We determined optimal muscle length (Lo), the length that generates maximal twitch force, and used this length th roughout the protocol. Lo was found by systematically adjusting the length of th e muscle while stimulating it with single supramaximal (~150%) twitches and recording the force generated. Lo was measured (in cm) using calipers. Peak isometric tetanic tension was measured from a series of three contractions with 2 min of recovery between contract ions. The force-frequency relationship was studied by stimulating the muscle stri ps at 15, 30, 60, 100, 160, and 200 Hz (120 V). Each stimulus was applied for 500 ms, and adjacent stimulus trains were separated by 2 min of rest. Diaphragmatic fatigability was assess ed by monitoring the decrease in force development over time. Each muscle strip wa s stimulated by unfused tetanic contractions (30 Hz, 250 ms) for 30 min. The duty cycle, or time of muscle cont raction compared to muscle rest, was 12.5%. Tension was meas ured at 0, 1, 2, 5, 10, 15, 20, 25, and 30 min. Fatigue resistance was assessed by the percentage of initial force maintained at the end of the 30 min protocol. After all contractile measurements were made, the diaphragm strip was removed from the organ bath. The rib, central tendon, and excess fat and connective tissue were removed from the strip, which was then blotted and weighed. Forces generated were normalized to muscle strip cross-sectional area (CSA).

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32 Protein Degradation To measure total protein degr adation, the release of tyrosine into the incubation medium was measured. Two strips were cu t from the midcostal diaphragm (~ 40 mg each). These strips were secured at resti ng length in separate baths containing KrebsRinger bicarbonate solution, wh ich was supplemented with 5 mM glucose, insulin (1 unit/ml), 0.17 mM leucine, 0.10 mM isoleuci ne, and 0.20 mM valine to improve protein balance, and 5 mM cycloheximide to inhibit protein synthesis. Di aphragm strips were maintained at resting lengths by securing both ends to a solid plexiglass rod. The medium was continuously gassed with 95% O2 5% CO2. Temperature was maintained at 37oC. Muscle strips were preincuba ted for 30 min, and then fresh medium was added for a 2 h incubation. After this incuba tion, the strips were removed, blotted, and weighed. For measurement of tyrosine re lease, the medium from each bath was aliquoted into microcentrifuge tubes. The a liquots were stored at –20oC until analysis of tyrosine concentration. Tyrosine in the medium was assayed sp ectrofluorometrically by the method of Waalkes and Udenfriend (1957) with some modification. Two hundred l of incubation medium was diluted with 800 l dH2O in a glass tube. To th is, 0.5 ml of 1-nitroso-2naphthol reagent (0.1 g 1-nitros o-2-naphthol in 100 ml of 95% methanol) and 0.5 ml of nitric acid reagent (24.5 ml of 20% nitric acid and 0.5 ml of 2.5% NaNO2) were added. The tubes were shaken to mix, and incubated in a water bath at 55oC for 30 min. After cooling for 15 min, 5.0 ml of ethylene dichlo ride was added to extract the unchanged nitrosonaphthol reagent. The tubes were centrifuged for 15 min at 2500 x g. One ml of the supernatant was transferred to a quartz cuvette and read in a spectrophotofluorometer.

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33 The tyrosine derivative was excited at 460 uM and measured at 570 uM. Standards were prepared using L-tyrosine (Sigma). 20S Proteasome Activity The chymotrypsin-like activity of the 20S proteasome was measured fluorometrically as the release of AMC from the synthetic substrate Suc-LLVY-AMC. Approximately 50 mg of midcostal diaphr agm tissue was homogenized (glass-on-glass) in a homogenizing buffer containing 50 mM Tris base, 1 mM EDTA, 1 mM EGTA, 1 M Pepstatin-A, 50 M E-64, and 10% glycer ol. This homogenate was centrifuged at 1500 x g for 10 min at 4oC, and the supernatant was then centrifuged at 10,000 x g for 10 min at 4oC. The remaining supernatant was centrifuged at 100,000 x g in an ultracentrifuge for 1 h at 4oC to separate the proteasomal fraction. The resulting supernatant fraction was used to measure pr otein content using the Bradford method, and to measure proteasome activity. Ten g of prot ein was reacted with the synthetic peptide substrate for chymotrypsin-like activity in a reaction mixture cont aining 50mM Tris-HCl, 1 mM DTT, and 5 mM MgCl2. One aliquot from each sample was incubated with an inhibitor of the chymotrypsin-like proteasom al activity, lactacysti n, while the other was not. Samples were incubated for 30 min at 37oC before the addition of substrate. The change in fluorescence was measured at an excitation wavelength of 380 nM and emission of 460 nM. The difference between th e activities of the proteasome with and without inhibitor was used as the proteasome activity. Total and Non-protein Thiols As an indicator of oxidative stress, we measured total thiol and non-protein thiol groups in diaphragm homogenate. Diaphr agm tissue was homogenized in 0.02 M EDTA on ice and centrifuged at 1500 rpm for 10 min at 5 oC. Twenty-five l of homogenate was

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34 incubated with 75 l 0.2 M Tris buffer (p H 8.2), 395 l methanol, and 5 l 0.01 M DTNB for 35 min on a bench-top rotator. Samples we re centrifuged at room temperature for 15 min at 3,000 x g. Two hundred l of supernatan t was loaded per microplate well (3 wells per sample) and read at a wavelength of 414 nm. A standard curve was generated using glutathione (GSH; Sigma) in 0.02 M EDTA. Non-protein thiols were measured by incubating 350 l homogenate with 350 l 1% metaphosphoric acid for 15 min on a benc h-top rotator to pr ecipitate proteins. Samples were centrifuged at room temperature for 15 min at 3,000 x g. Three hundred l supernatant was then incubated with 200 l 0.4 M Tris buffer (pH 8.9) and 25 l 0.01 M DTNB. Tubes were mixed for 10 min and read against GSH standards in a microplate reader at 414 nm. Limitations We did not record electromyographic (E MG) activity of the mechanically ventilated diaphragms to ensure that mu scle activation was completely suppressed. Powers et al. (2002) performed preliminar y experiments where wire electrodes that measure EMG activity were placed in the costal diaphragm of 4 animals during 24 h of controlled MV. No electrical activity was meas ured in any of these animals during the 24 h procedure. Le Bourdelles and colleagues (1994) did not find EMG activity in the rat diaphragm ventilated for 2 days. Therefore, we assumed that diaphragmatic contractions did not occur during 12 h of controlled MV. The mortality rate of the SBT group was higher than other groups (~70%). As a result, we were only able to include 3 an imals that survived the 12-h spontaneous breathing protocol while receiving Trolox. This made statistical an alyses difficult, and conclusions yet uncertain with this group.

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35 Vertebrate Animals Female Sprague-Dawley rats were used in this research. This study required removal of the diaphragm musc le for analysis, and therefor e prevented the use of human subjects. Sprague-Dawley rats were select ed since our lab, as well as previous researchers, has successfully used them as s ubjects in MV studies. The MV experiments were supervised by research as sistants who have experience w ith short-term MV in rats. Statistical Analysis This experiment was designed to test the hypotheses that Trolox supplementation during mechanical ventilation would alter diaphragm contractil e dysfunction, protein degradation, and oxidative stress. A 4 x 6 (g roup x stimulation frequency) ANOVA with repeated measures on stimulation frequency was used to analyze the force-frequency data. Likewise, a 4 x 9 (group x time) ANOVA w ith repeated measures on the time factor was used to analyze data from the fatigue protocol. Where significant differences were found, Tukey’s HSD test was implemented pos t hoc. ANOVAs were used to examine differences between groups for the remaini ng dependent variables. Independently, the effects of anesthesia were compared using a Student’s t-test on SBS and CON group data. Significance was established at p<0.05.

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36 CHAPTER 4 RESULTS Systemic and Biologic Responses to Treatment The MV protocol did not significantly change body mass for any of the groups (Table 1), indicating that our schedule of nutrition and rehydration was adequate. The ratio of total costal diaphragm mass to final body mass was not si gnificantly different between the 5 groups (p=0.501). There were no signs of infection in a ny animals, and only 1 MVS animal was eliminated from the study due to evidence of barotrauma to the lungs on post-mortem examination. Systolic blood pressure was ma intained at 70-110 mmHg in all groups, and arterial pH, PO2, and PCO2 were maintained within physiological ranges for both MV groups. The SB animals were mildly hypoxic, hy percapnic, and acidotic as expected due to the anesthesia. Body temp erature was kept at 37 + 1oC during the 12-hour protocol. Effects of Anesthesia on Diaphragm Contractile Properties The maximal tetanic force was not different between the SBS and CON groups (25.09 + 0.41 N/cm2 vs. 25.33 + 0.50 N/cm2, respectively). Likewise, the force-frequency curves and fatigue data were similar between these two groups (Fi gures 1, 2, and 3), and contractile parameters did not differ (Table 3). Thus, 12 hours of sodium pentobarbital anesthesia did not affect in vitro cont ractile properties of the diaphragm. Effects of Mechanical Ventilati on on Contractile Properties Twelve hours of controlled MV reduced ma ximal tetanic force production by ~17% (21.00 + 0.71 N/cm2 vs. 25.43 + 0.50 N/cm2 in CON animals). The force-frequency curve

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37 of the MVS group was shifted downward and to the right of the C ON group (Figure 1). This indicates a reduction in force generation at all stimul ation frequencies tested. The fatigue protocol produced curves of similar shape for all groups (F igure 2), but the MVS group generated a significantly lower amount of force compared to CON, SBS, and MVT groups where indicated. When the fatigue data are expressed as percent of initial force (Figure 3), there are no significant differences between the 4 groups at any time point (p= 0.230). One-half relaxation time ( RT) of maxima l twitch was significantly shorter in the MVS group compared to CON, while time to peak tension ( TPT), rate of force development, and rate of relaxation were not different for either maximal twitch or maximal tetanic forces (Table 3). Effects of Trolox on Contractile Properties Trolox supplementation during 12 hours of MV completely attenuated the loss of maximal force generation. The MVT group was not significantly diffe rent from CON at any stimulation frequency tested (Figur e 1). Animals receiving Trolox during MV maintained a greater force generating ability following the fatigue protocol compared to the unsupplemented MVS group (Figure 2). Tr olox during MV significantly prolonged the rate of relaxation of maximal twitch co mpared to CON, but did not affect other contractile parameters (Table 3). Twelve hours of Trolox infusion without MV (i.e. the SBT group) greatly increased subject mortality rate. Only three SBT an imals survived the treatment. Further, diaphragms from these animals showed impaired force production compared to CON. The force-frequency curve of the SBT group was shifted downward and to the right of the CON group and closely resemble s the MVS group (Figure 1). Trolox

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38 supplementation during spontaneous breathing also significantly reduced TPT of maximal tetanic force compared to CON animals. Protein Degradation Twelve hours of controlled MV significantly elevated total in vitro protein degradation (+105%), as measur ed by the release of free tyrosine, compared to CON (Figure 4). However, protein degradation of the MVT group ( 16% increase compared to CON) was not significantly different from CON (p=0.797). There were no significant differences in protein degradation between CON and SBS groups (p=0.351). 20S Proteasome Activity The chymotrypsin-like activity of the 20S proteasome was significantly increased in the MVS group compared to the CON group (+76%) (Figure 5). Trolox attenuated the MV-induced increase in proteasome activ ity (+26% compared to CON, p=0.647). Oxidative Stress Protein carbonyls and lipid hydr operoxides, two indicators of oxidative stress, were not different between CON, MVS, and MV T groups. However, total thiols were significantly lower in the MVS group as compared to the CON group (134.83 + 6.90 nmol/mg protein vs. 157.34 + 6.45 nmol/mg,respectively). Similarly, non-protein thiols were significantly lower in the MVS group compared to the CON group (28.02 + 1.62 nmol/mg vs. 42.14 + 1.51 nmol/mg, respectively). But, Trolox supplementation during MV failed to prevent the loss of total and non-protein thiol groups.

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39 Table 4-1. Body and Diaphragm Weights of Control, Spontaneously Breathing, and Mechanically Ventilated Animals CON SBS MVS SBT MVT Initial body mass (g) 264.38 + 5.16 276.88 + 5.29 282.25 + 3.33 285.67 + 2.33 300.13 + 6.37*† Final body mass (g) 264.38 + 5.16 280.56 + 5.52 286.38 + 3.18* 292.00 + 2.00 306.75 + 6.42*† Total costal diaphragm mass (mg) 532.57 + 15.64 567.69 + 12.86 601.86 + 14.80 610.63 + 27.98 632.98 + 10.74 Total costal diaphragm mass/body mass‡ (mg/g) 2.014 + 0.04 2.025 + 0.04 2.100 + 0.04 2.091 + 0.09 2.067 + 0.04 Definition of abbreviations: CON = control animals; SBS = spontan eously breathing animals receiving saline; MVS = mechanically ventila ted animals receiving saline; SBT = spontaneously breathing animals receiving Trolox; MVT = mechanically ventilated animals receiving Trolox. Values represent means + SEM Significantly different from CON group, p<0.05. † Significantly different from SBS group, p<0.05. ‡ Mass values expressed as milligrams pe r gram of body mass were normalized to postexperiment body mass values. Table 4-2. Maximal Isometric Twitch and Te tanic Force of Control, Spontaneously Breathing, and Mechanically Ventilated Animals CON SBS MVS SBT MVT Maximal isometric twitch force (N/cm2) 7.24 + 0.14 6.79 + 0.30 5.65 + 0.28*†‡ 5.94 + 0.55 7.04 + 0.25 Maximal isometric tetanic force (N/cm2) 25.43 + 0.50 25.09 + 0.41 21.01 + 0.71*†‡ 21.54 + 1.40*† 25.49 + 0.50 Definition of abbreviations: CON = control animals; SBS = spontan eously breathing animals receiving saline; MVS = mechanically ventila ted animals receiving saline; SBT = spontaneously breathing animals receiving Trolox; MVT = mechanically ventilated animals receiving Trolox. Values represent means + SEM Significantly different from CON group, p<0.05. † Significantly different from SBS group, p<0.05. ‡ Significantly different from MVT group, p<0.05.

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40 Table 4-3. Contractile Parameters of Maxima l Isometric Twitch and Tetanic Forces of Control, Spontaneously Breathing, and Mechanically Ventilated Animals CON SBS MVS SBT MVT TWITCH TPT 0.018 + 0.000 0.018 + 0.001 0.017 + 0.000 0.017 + 0.000 0.018 + 0.000 RT 0.044 + 0.001 0.040 + 0.001 0.034 + 0.003* 0.037 + 0.002 0.04 + 0.001 + dp/dt 332.29 + 27.368 420.97 + 31.176 415.85 + 57.784 418.82 + 94.619 459.52 + 18.567 dp/dt -149.26 + 9.247 -177.84 + 9.696 -207.93 + 28.890 -179.61+ 34.138 -204.21 + 7.845* TETANIC TPT 0.064 + 0.002 0.062 + 0.001 0.056 + 0.003 0.053 + 0.003* 0.059 + 0.002 RT 0.064 + 0.002 0.062 + 0.003 0.062 + 0.003 0.061 + 0.001 0.065 + 0.002 + dp/dt 423.71 + 34.738 527.67 + 41.175 515.38 + 95.654 518.80 + 93.776 570.18 + 20.668 dp/dt -544.55 + 50.860 -723.04 + 36.225 -703.53 + 157.300 -658.41 + 94.133 -740.20 + 12.091 Definition of abbreviations: CON = control animals; SBS = spontan eously breathing animals receiving saline; MVS = mechanically ventila ted animals receiving saline; SBT = spontaneously breathing animals receiving Trolox; MVT = mechanically ventilated anim als receiving Trolox; TPT = time to peak tension; RT = relaxation time; +dp/dt = rate of force development; -dp/dt= rate of force relaxation. Values represent means + SEM Significantly different from CON group, p<0.05. † Significantly different from SBS group, p<0.05. ‡ Significantly different from MVT group, p<0.05.

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41 Stimulation Frequency (Hz) 255075100125150175200Force (N/cm2) 10 12 14 16 18 20 22 24 26 CON SBS MVS MVT *,+,#*,+,#*,+,#*,+,#*,+,#*,+,# Figure 4-1. Force-frequency res ponses of control (CON), spont aneously breathing (SBS), mechanical ventilation (MVS), and m echanical ventilation animals receiving Trolox (MVT). Values represent means + SEM. Significantly different from CON group, p<0.05. + Significantly different from SBS group, p<0.05. # Significantly different from MVT group, p<0.05.

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42 Time (min) 05101520253035Force (N/cm2) 6 8 10 12 14 16 18 20 CON SBS MVS MVT *,+,#*,+,#*,#*,#*,+,#*,+,#*,+,#*,+,#*,+,#*,+,#*,+,# Figure 4-2. Responses of in vitro diaphragm strips from control (CON), spontaneously breathing (SBS), mechanical ventilati on (MVS), and mechanical ventilation animals receiving Trolox (MVT) to a 30-min fatigue protocol. Values represent means + SEM. Significantly different from CON group, p<0.05. + Significantly different from SBS group, p<0.05. # Significantly different from MVT group, p<0.05.

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43 Time (min) 05101520253035Percent of Initial Force 20 40 60 80 100 CON SBS MVS MVT Figure 4-3. Percent of initi al force maintained by in vitro diaphragm strips from control (CON), spontaneously breathing (SBS), mechanical ventilation (MVS), and mechanical ventilation animals receivi ng Trolox (MVT) after a 30-min fatigue protocol. Values represent means + SEM. Significantly different from CON group, p<0.05. + Significantly different from SBS group, p<0.05. # Significantly different from MVT group, p<0.05.

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44 Group Tyrosine ( mol/g/2 h) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 CON SBS MVS MVT *,+,# Figure 4-4. Total in vitro diaphragmatic protein degradati on as measured by the rate of tyrosine release from control (CON) spontaneously breathing (SBS), mechanical ventilation (MVS), and m echanical ventilation animals receiving Trolox (MVT) during a 2-hour incubation. Values represent means + SEM. Significantly different from CON group, p<0.05. + Significantly different from SBS group, p<0.05. # Significantly different from MVT group, p<0.05.

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45 Group 20S Proteasome Activity ( mol/min/mg protein) 0.0 0.2 0.4 0.6 0.8 CON SBS MVS MVT Figure 4-5. Chymotrypsin-like activity of the 20 S proteasom e in diaphragm tissue from control (CON), spontaneous ly breathing (SBS), mechanical ventilation (MVS), and mechanical ventilati on animals receiving Trolox (MVT). Values represent means + SEM. Significantly different from CON group, p<0.05.

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46 Group Total Thiols (nmol/mg protein) 100 120 140 160 180 200 CON SBS MVS MVT * Figure 4-6. Total thiol concentration in diaphragm tissue fr om control (CON), spontaneously breathing (SBS), mechan ical ventilation (MVS), and mechanical ventilation anim als receiving Trolox (MVT). Values represent means + SEM. Significantly different from CON group, p<0.05.

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47 Group Non-Protein Thiols (nmol/mg protein) 10 20 30 40 50 60 CON SBS MVS MVT *,+,#*,+ Figure 4-7. Non-protein thiol concentration in diaphragm tissue from control (CON), spontaneously breathing (SBS), m echanical ventilation (MVS), and mechanical ventilation anim als receiving Trolox (MVT). Values represent means + SEM. Significantly different from CON group, p<0.05. + Significantly different from SBS group, p<0.05. # Significantly different from MVS group, p<0.05.

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48 CHAPTER 5 DISCUSSION Trolox Attenuates Mechanical Ventilation -induced Contractile Dysfunction and Proteolysis in the Rat Diaphragm:Introduction Weaning patients from a mechanical vent ilator is a serious clinical issue. Mechanical ventilation (MV) is characteristically used in the clinical setting to maintain alveolar ventilation in patients who are in capable of ventilation on their own. As such, MV is an important life-preserving measure, but removing patients from the ventilator can be difficult in many cases. As many as 20% of patients experi ence difficulty in weaning from the ventilator (Lemaire 1993). Weaning procedures account for more than 40% of total MV time in patients who ha ve difficulty weaning (Esteban 1994), suggesting that this is a se rious clinical issue. Our laboratory has reported that MV-induced diaphragmatic dysfunction is intrinsic to the muscle and increases in magnitude w ith increasing time on th e ventilator (Powers et al. 2002). However, the mechanism(s) behind MV-induced diaphragmatic atrophy and weakness remain unclear. Since oxidative stress has been link ed to reduced-use atrophy (Kondo et al. 1993), and protease-mediated pr otein degradation in unloaded locomotor muscle (Taillandier et al 1996), we examined markers of oxidative stress during MV. Eighteen hours of controlled MV elevated ma rkers of oxidative stress such as protein carbonyls and 8-isoprostanes (Shanely et al. 2002 ). This is significant because oxidized proteins are more prone to proteolytic a ttack and degradation (Dean 1997, Nagasawa et al. 1997). Indeed, 18 hours of MV is associated with an increase in protein degradation

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49 through increases in total calpain -like activity and 20S proteasome activity (Shanely et al. 2002). Therefore, it seems logical that di aphragmatic weakening during MV-induced unloading may be caused by oxidative damage leading to heightened proteolytic degradation. The purpose of this study was to determ ine whether supplementation with Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) during 12 hours of controlled MV would attenuate diaphrag matic contractile dysfunction, reduce oxidative stress, and attenuate protein degradation. Trolox is a water-soluble vitamin E analog with antioxidant properties (Wu et al. 1990, Zeng et al. 1991, Walker et al. 1998). We hypothesized that Trolox would maintain re dox balance within the muscle by functioning as an antioxidant, and thereby prevent oxida tive stress, and subse quent proteolysis and contractile dysfunction. Materials and Methods Animals and Experimental Design Female Sprague-Dawley rats (~250 g) were obtained from Harlan (Indianapolis, IN). They were maintained on a 12-hour light:dark photoperiod and fed rat chow and water ad libitum prior to initiation of experiments. Animals were randomly assigned to one of five groups: (1) Controls receiving acute anesthesia and no further intervention (CON, n=8) (2) 12 hours of mechanical ventil ation (MVS, n=8), (3) 12 hours of MV and Trolox infusion (MVT, n=8), (4) 12 hours of an esthesia and spontaneous breathing (SBS, n=8), and (5) 12 hours of anesthesia and s pontaneous breathing with Trolox infusion (SBT, n=3). All procedures were approved by th e University of Florida Animal Care and Use Committee.

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50 Control Animal Protocol The control animals (CON) were free of intervention before removal of the diaphragm for measurements. These animals re ceived an acute intraperitoneal injection of sodium pentobarbital (65 mg/kg body weight ). When a surgical plane of anesthesia was reached, the diaphragms were removed for measurement of in vitro contractile properties and tyrosine release, and rema ining muscle was weighed and frozen for biochemical assays. Mechanical Ventilation Protocol Animals in the mechanically ventilated groups (MVS and MVT) were given an intramuscular injection of gl ycopyrollate (0.04 mg/kg) to re duce respiratory secretions. Thirty minutes later, subjects were anesthe tized with an intraper itoneal injection of sodium pentobarbital (65 mg/kg). Upon reaching a surgical plane of anesthesia (no ocular response, no hindlimb withdrawal respons e), they were tracheostomized by an experienced lab technician and mechanically ventilated with a volum e-cycled ventilator (Harvard Apparatus). The tidal volume was es tablished at ~1 ml/100 g body weight with a respiratory rate of 80 breaths/min. Positive end-expiratory pressure of 1 cm H2O was used for all ventilated animals. Throughout the MV period, heart act ivity, blood pressure, and core temperature were monitored. A lead II ECG displayed electr ical activity of the heart. A catheter was placed in the carotid ar tery for constant blood pressure readings and arterial blood sampling. Core temperature wa s monitored with a rectal thermometer and adjustments were made to maintain body temperature at 37 1oC with a re-circulating heating blanket. A catheter was placed in the jugular vein for the infusion of sodium pentobarbital (~10 mg/kg/h) and Trolox (Fluka). Consta nt supervision was provided for the rats

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51 throughout the MV period. This included expressing the bladder, removing airway mucus, providing enteral nutrition, monitoring anesthesia rate, ro tating the animals, lubricating the eyes, and infusing saline to maintain hydration status. To reduce airway secretions, glycopyrollate (0.04 mg/kg) was injected intramuscularly every 2 hours. In the MVT group, the jugular vein was cannulated first. A priming dose of Trolox (20 mg/kg) was infused over a 5 min period. Twenty min later, MV was started, along with the constant infusion of Trolox at a rate of 5 mg/kg/h. At the end of the 12-hour experimental pe riod, all rats were killed by injection of sodium pentobarbital (50 mg/kg) and the di aphragm was removed for immediate analyses of contractile function and protein degrada tion as described belo w. Remaining costal diaphragm tissue was dissected, weighed, frozen, and stored for biochemical analyses. Spontaneous Breathing Protocol Spontaneously breathing (SB) animals were anesthetized in th e same manner as MV animals and received sham surgeries. Th ese animals were included in the study to determine whether long-term anesthesia (s odium pentobarbital) im pairs diaphragmatic contractile function. A tube was inserted into the trachea, and these animals were maintained on a surgical plane of anesthes ia for the 12-hour period while continuing to breathe on their own. The carotid artery and jugular vein were cannulated, and sodium pentobarbital was infused for the 12 hours. However, these animals were not mechanically ventilated. The SBT group received the same dose of Trolox as the MVT group (20 mg/kg priming dose, 5 mg/kg/h constant infusion) for the 12-hour experimental period. The same general care was provided for these animals as for MV animals.

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52 Contractile Measurements The entire diaphragm with the supporting ribs and central tendon was removed and placed in a dissecting chamber containing a Krebs-Hensleit solution aerated with 95% O2-5% CO2 gas. The entire crural diaphragm wa s removed and discarded. A strip was cut from the midcostal region including the cen tral tendon and the rib. This was secured vertically in an organ bath maintained at 24oC between two Plexiglass clamps. The muscle strip was placed between two platinum field electrodes connected to an isometric force transducer (model FT-03, Grass Instrume nts, Quincy, MA). This strip was mounted on a micrometer to allow for muscle length adjustment. A 15 min equilibration period in the bath preceded all data collection. Duri ng this time, the remaining diaphragm muscle was dissected and sectioned. All sections we re weighed, frozen in liquid nitrogen, and stored at –80oC for further analyses. We determined optimal muscle length (Lo), the length that generates maximal twitch force, and used this length throughout the protocol. Lo was found by systematically adjusting the length of the muscle while s timulating it with single supramaximal (~150%) twitches and recording the force generated. Lo was measured using calipers. The force-frequency relationship was studied by stimulating the muscle strips at 15, 30, 60, 100, 160, and 200 Hz (120 V). Each stim ulus was applied for 500 ms, and adjacent stimulus trains were separated by 2 mi n of rest. Peak isometric tetanic tension was determined from these measurements. Diaphragmatic fatigability was assess ed by monitoring the decrease in force development over time. Each muscle stri p was stimulated with unfused tetanic contractions (30 Hz, 250 ms) for 30 min. The duty cycle, or time of muscle contraction compared to muscle rest, was 12.5%. Tens ion was measured at 0, 1, 2, 5, 10, 15, 20, 25,

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53 and 30 min. Fatigue resistance was assessed by the percentage of initial force maintained at the end of the 30 min protocol. After a ll contractile measurements were taken, the diaphragm strip was removed from the organ bath. The rib, central tendon, and excess fat and connective tissue were removed from the strip, which was then weighed. Forces generated were normalized to muscle strip cr oss-sectional area (CSA), calculated from strip weight and length at Lo. Protein Degradation To measure total in vitro protein degradation, the releas e of free tyrosine into the incubation media was assayed. The rationale for th is technique is that tyrosine is neither synthesized nor degraded by skeletal muscle, making it an ideal marker of total muscle protein breakdown (Tischler et al. 1982). Tw o strips were cut from the midcostal diaphragm (~ 40 mg each). These strips were secured at resting length in separate baths containing Krebs-Ringer bicarbonate solu tion, which was supplemented with 5 mM glucose, insulin (1 unit/ml), 0.17 mM leuc ine, 0.10 mM isoleucine, and 0.20 mM valine to improve protein balance, and 5 mM cycl oheximide to inhibit protein synthesis. Diaphragm strips were maintained at res ting length by securing bot h ends to a solid plexiglass support. Muscle strips were susp ended vertically in the organ bath. The medium was continuously gassed with 95% O2-5% CO2, and temperature was maintained at 37oC with a recirculating water bath. Afte r a 30-min pre-incubation period, the media was drained, and fresh media was quickly a dded for a 2-hour incubation. Rates of total protein breakdown were measured by assa ying tyrosine release into the medium according to the spectrofluorometric me thod of Waalkes and Udenfriend (1957).

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54 20 S Proteasome Activity The in vitro chymotrypsin-like activity of the 20S proteasome was measured fluorometrically by following the release of free AMC from the synthetic substrate SucLeu-Leu-Val-Tyr-AMC (Affiniti Research) usin g techniques described by Stein et al. (1996). Briefly, ~50 mg of midcostal dia phragm tissue was homogenized in buffer containing 50 mM Tris base, 1 mM EDTA, 1 mM EGTA, 1 M Pepstatin-A, 50 M E64, and 10% glycerol. After in itial centrifugations, the s upernatant was collected and centrifuged at 100,000 x g in an ultracentrif uge for 1 hour at 4oC. This supernatant fraction was used to measure protein conten t using the Bradford method (Bradford 1976), and to measure 20S proteasome activity as follows. Ten g of protein was reacted with the synthetic peptide substrate for chymot rypsin-like activity (Suc-LLVY-AMC) in a reaction mixture containing 50mM Tr is-HCl, 1 mM DTT, and 5 mM MgCl2. One aliquot from each sample was incubated with an inhibitor of the chymotrypsin-like proteasomal activity, lactacystin (Boston Biochem), while the other wa s not incubated with the inhibitor. Samples were incubated for 30 min at 37oC before the addition of substrate. The change in fluorescence was measured at an excitation wavelength of 380 nM and emission of 460 nM. The difference between th e activities of the proteasome with and without inhibitor was used as the 20S proteasome activity. Statistical Analysis Comparisons between groups were made by a one-way ANOVA. Where significant differences were found, Tukey’ s HSD test was implemented post hoc Independently, the effects of anesthesia were compared using a Student’s t-test on SBS and CON group data. Significance was established a priori at p<0.05.

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55 Results Systemic and Biologic Responses to Treatment The MV protocol did not significantly ch ange body mass for any of the 5 groups (Table 4-1), indicating that our schedule of nutrition and rehydration was adequate. The ratio of total costal diaphragm mass to final body mass was not si gnificantly different between the 5 groups (p=0.501). There were no signs of infection in a ny animals, and only 1 MVS animal was eliminated from the study due to evidence of barotrauma to the lungs on post-mortem examination. Systolic blood pressure was ma intained at 70-110 mmHg in all groups, and arterial pH, PO2, and PCO2 were maintained within physiological ranges for both MV groups. The SB animals were mildly hypoxic, hy percapnic, and acidotic as expected due to the anesthesia. Body temp erature was kept at 37 + 1oC during the 12 hour protocol. Effects of Anesthesia on Di aphragm Contractile Properties The maximal tetanic force was not different between the SBS and CON groups (25.09 + 0.41 N/cm2 vs. 25.33 + 0.50 N/cm2, respectively). Likewise, the force-frequency curves and fatigue data were similar between these two groups (Figures 4-1, 4-2, and 43), and contractile parameters did not di ffer (Table 4-3). Thus, 12 hours of sodium pentobarbital anesthes ia did not affect in vitro contractile properties of the diaphragm. Effects of Mechanical Ventilati on on Contractile Properties Twelve hours of controlled MV reduced ma ximal tetanic force production by ~17% (21.00 + 0.71 N/cm2 vs. 25.43 + 0.50 N/cm2 in CON animals). The force-frequency curve of the MVS group was shifted downward and to the right of the CON group (Figure 4-1). This indicates a reduction in force generation at all stimul ation frequencies tested. The fatigue protocol produced curves of similar shape for all groups (Figure 4-2), but the

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56 MVS group generated a significantly lower amount of force compared to CON, SBS, and MVT groups where indicated. When the fatigue data are expressed as percent of initial force (Figure 4-3), there are no significant di fferences between the 5 groups at any time point (p= 0.230). One-half relaxation time ( RT) of maxima l twitch was significantly shorter in the MVS group compared to CON, while time to peak tension ( TPT), rate of force development, and rate of relaxation were not different for either maximal twitch or maximal tetanic forces (Table 4-3). Effects of Trolox on Contractile Properties Trolox supplementation during 12 hours of MV completely attenuated the loss of maximal force generation. The MVT group was not significantly diffe rent from CON at any stimulation frequency tested (Figur e 4-1). Animals receiving Trolox during MV maintained a greater force generating ability following the fatigue protocol compared to the unsupplemented MVS group (Figure 4-2). Tr olox during MV signifi cantly prolonged the rate of relaxation of maximal twitch co mpared to CON, but did not affect other contractile parameters (Table 4-3). Twelve hours of Trolox infusion without MV (i.e. the SBT group) greatly increased subject mortality rate. Only three SBT an imals survived the treatment. Further, diaphragms from these animals showed impaired force production compared to CON. The force-frequency curve of the SBT group was shifted downward and to the right of the CON group and closely resembles the MVS group (data not shown). Trolox supplementation to SB’s also significantly reduced TPT of maximal tetanic forces compared to CON animals (Table 4-3).

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57 Protein Degradation Twelve hours of controlled MV significantly elevated total in vitro protein degradation (+105%), as measur ed by the release of free tyrosine, compared to CON (Figure 4-4). However, protei n degradation of the MVT group (16% increase compared to CON) was not significantly different fr om CON (p=0.797). There were no significant differences in protein degradation between CON and SBS groups (p=0.351). 20S Proteasome Activity The chymotrypsin-like activity of the 20S proteasome was significantly increased in the MVS group compared to the CON gr oup (+76%) (Figure 4-5) Trolox attenuated the MV-induced increase in proteasome activity (+26% compared to CON, p=0.647). Discussion Major Findings The major findings of this study are: 1) Trolox supplementation during 12 hours of controlled MV attenuates di aphragmatic contractile dysfunction and whole muscle proteolysis; 2) 12 hours of anes thesia and spontaneous breathing do not affect contractile function or protein degradation within the diaphragm; 3) Prot eolysis is elevated during 12 hours of MV in part due to increased chymot rypsin-like activity of the 20S proteasome, and 4) Trolox supplementation during normal spontaneous breathing shifts redox balance to a reductive state which actually impairs diaphragmatic function. MV and Diaphragmatic Dysfunction These data support our previous studies (Powers et al. 2002, Sh anely et al. 2003) that show a reduction in maximal force produc tion with prolonged MV These data agree with other MV studies with rats (Le Bourdelles et al. 1994 ), baboons (Anzueto et al. 1997), piglets (Radell et al 2002), and rabbits (Sassoon et al. 2002, Capdevila et al.

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58 2003). In the present study, ma ximal tetanic tension was decreased ~17% with 12 hours of controlled MV. Le Bourdelles et al (1994) demonstrated that 48 hours of MV significantly reduced diaphragmatic force w ithout altering protein concentrations or enzyme activities. Anzueto et al (1997) found a decrease in transdiaphragmatic pressure and diaphragmatic endurance after 11 days of MV in baboons. However, the use of longlasting neuromuscular blockers in this study may have affected diaphragm responses. We have shown that the degree of diaphragmatic dysfunction is proportional to the length of time of MV (Powers et al. 2002). Radell and colleagues (2002) demonstrated that diaphragmatic dysfunction induced by 5 days of MV in a piglet model is not asso ciated with alterations in nerve function or neuromuscular transmission. With respect to muscle fiber composition, Capdevila et al. (2003) found significant atrophy of type IIa and IIb fibers, with no changes in type I fibers, in the rabbit diaphragm ventilated for 51 hours. In the rat, 4 days of MV caused a decrease in the percentage of type I fibers, a nd an increase in hybrid fibers co-expressing type I and II MHC (Yang et al. 2002). Also, Shanely and co lleagues (2002) found atrophy of all fiber types that was greatest in type II fibers after just 18 hours of MV. Finally, significant myofibril damage in the diaphragm is evident after MV (Sassoon et al. 2002).Together, these altera tions in diaphragmatic structure may lead to the weakness characteristic of a mechanically ventilated diaphragm muscle. In the present study, Trolox completely atte nuated the decrease in maximal specific tension that occurs during MV. These anim als were not different from CON or SBS animals with respect to maximal tetanic tens ion, maximal twitch tension, force-frequency relationships, and fatigue data.

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59 Oxidative Stress and Trolox Unloaded skeletal muscle is susceptible to oxidative stress duri ng periods of disuse (Kondo et al. 1993a, 1993b). During MV, the dia phragm muscle is both unloaded and passively shortened (Racz et al. 2003). These stimuli ar e likely to stimulate the unloading-induced atrophy typical of prolonged MV. Pr evious studies from our laboratory have measured an increase in pr otein oxidation and lipid peroxidation with 18 hours of MV (Shanely et al. 2002), and as early as 6 hours of MV (Zergeroglu et al. 2003). In the present study, we measured a decrease in tota l and non-protein thiols with 12 hours of MV consistent with oxidative st ress (data not shown). However, Trolox did not prevent the loss of thiol groups during MV. It may be that specific proteins, such as myosin, are preferentially spar ed from oxidation while others are not. If true, we were not able to detect these proteins in crude homogenate. However, we postulate that Trolox is functioning as an antioxidant to prevent oxida tive stress. Several studies have shown that Trolox reduces oxidative stress induced by cumene hydroperoxide (Persoon-Rothert et al. 1990), methylmercury intoxicati on (Usuki et al. 2001), and ot her oxyradicals (Wu et al. 1990, Wu et al. 1991, Zeng et al. 1991, Walker et al. 1998). Wu and colleagues (1990) have even demonstrated antioxidant actions of Trolox in three human cell types exposed to oxyradicals. While we were unable to cl early demonstrate the prevention of oxidative stress with Trolox, it is likely that Trolox is acting as an antioxidant to scavenge reactive oxygen species produced in the diaphr agm during 12 hours of controlled MV. MV and Proteolysis Twelve hours of controlled MV significan tly increased (+105 %) the release of tyrosine from in vitro diaphragm strips. This agrees with a previous study from our laboratory reporting increased tyrosine release after 18 hour s of MV (Shanely et al.

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60 2002). Tyrosine release is freque ntly used as an indicator of total protein degradation (Lowell et al. 1986) since tyrosine is neither synthesized nor degraded by skeletal muscle. In our previous 18-hour MV study, we dete rmined that total calpain-like and 20S proteasome activities were elev ated, indicating a contributio n of these two proteolytic pathways to diaphragmatic proteoly sis during MV (Shanely et al. 2002). Importantly, Trolox attenuated the increase in total protein degradation induced by MV (Figure 4-4). Likewise, the chymotryps in-like activity of the 20S proteasome was elevated during MV, but this increase wa s prevented with Trolox (Figure 4-5). Many studies indicate that the prot easome is responsible for ~70-80% of the increased cellular protein degradation following an oxidative st ress (Grune et al. 1995, Grune et al. 1996, Grune and Davies 1997). More specifically, it appears the 20S proteasome is responsible for the degradation of oxidized proteins sin ce the 26S proteasome is inhibited/inactivated by oxidative stress (Reinheckel et al. 1998) Recognition of exposed hydrophobic patches is the proposed mechanism by which the prot easome selectively degrades oxidatively modified proteins (Grune et al. 1997). Oxidative damage to a protein leads to partial unfolding and exposure of normally shielded internal hydrophobic patches that are recognized by the proteasome, which cataly zes the degradation of that protein. The diaphragmatic atrophy and contractil e dysfunction that occur with prolonged MV are likely the result of increased oxidati ve modification of proteins, leading to proteolytic attack and degrad ation. This loss of protein, es pecially contractile protein, would result in atrophy and decreases in maxi mal force production. Clin ically, this would manifest as difficulty wean ing from the ventilator.

PAGE 71

61 Critique of Experimental Model It was necessary to use an animal model due to the invasive nature of this study. The rat was selected due to the similarities in anatomy and function of the rat and human diaphragms. Also, controlled MV, which is not as common clinically as pressure assist MV, was used because of the rapid onset of diaphragmatic atrophy characteristic of controlled MV. Prolonged anesthesia is known to lead to hypoxia, hypercapnia, and mild acidosis due to the reduced ventilatory drive in an anesthetized anim al (Powers et al. 2002). While these effects could theoretically affect in vitro diaphragm function, our data demonstrate that sodium pentobarbital an esthesia does not impact skeletal muscle function in vitro A comparison of the CON and SBS data indica te no significant differences in force generation, total proteolysis, or proteasome activity. Thus, the diaphragmatic dysfunction and proteolysis induced by MV were not caused by prolonged sodium pentobarbital use. We attempted to include a group to control for Trolox during spontaneous breathing (SBT group). However, only 3 animal s from this group survived the entire 12 hours, and these were not included in statistica l analyses. It is not surprising that Trolox was harmful in these animals that were not exposed to oxidants because Trolox is a strong reductant. We hypothesize that Trolox shifted the redox ba lance to a reductive state in the spontaneously breathing animals that impair ed diaphragmatic function. Conclusions Our results support earlier c onclusions that short-term controlled MV leads to diaphragmatic contractile dysfunction and increa sed protein degradation. Our data clearly demonstrate that an antioxida nt, Trolox, effectively preven ted contractile impairments and proteolysis during MV. Ox idative damage and atrophy ar e implicated in MV-induced

PAGE 72

62 contractile deficits. Oxidative damage to pr oteins during MV likely increases proteolytic degradation, which would contribute to di aphragmatic weakness. Trolox effectively spares the unloaded diaphragm from contract ile dysfunction, oxidative stress, and protein degradation during 12 hours of controlled MV. However, it is not warranted during normal spontaneous breathing, and can actually cause contractile dysfunction under such conditions. Future studies aiming to prevent force losses, oxidative stress, and protein degradation during MV might examine another vehicle and route of Trolox administration. In addition, other antioxidants need to be tested. The use of an antioxidant such as Trolox may prove beneficial in the clinical setting where weaning difficulties are encountered due to diaphragmatic atrophy and w eakness. This is a se rious clinical issue that warrants further investigation.

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63 LIST OF REFERENCES Anzueto, A; JI Peters; MJ Tobin; R De Los Santos; JJ Seidenfeld; G Moore; WJ Cox; JJ Coalson. Effects of prolonged controlled mechanical ventilation on diaphragmatic function in healthy adult baboons. Crit Care Med 25: 1187-1190, 1997. Appell, HJ; JAR Duarte; JMC Soares. Suppl ementation of vitamin E may attenuate skeletal muscle immobilization atrophy. Sports Med 18: 157-160, 1997. Baracos, V and AL Goldberg. Maintenance of normal length improves protein balance and energy status in isolated rat skeletal muscles. Am J Physiol 251: C588-C596, 1986a. Baracos, V; RE Greenberg; AL Goldberg. Infl uence of calcium and ot her divalent cations on protein turnover in rat skeletal muscle. Am J Physiol 250: E702-E710, 1986b. Berg, HE; L Larsson; PA Tesch. Lower limb sk eletal muscle functi on after 6 weeks of bed rest. J Appl Physiol 82(1): 182-188, 1997. Bernard, N; S Matecki; G Py; S Lopez; J Mercier; X Capdevila. Effects of prolonged mechanical ventilation on respiratory mu scle ultrastructure and mitochondrial respiration in rabbits. Int Care Med 29: 111-118, 2003. Booth, FW. Time course of muscular atrophy during immobilization of hindlimbs in rats. J Appl Physiol 43(4): 656-661, 1977. Bradford, MM. A rapid and sensitive method fo r the quantitation of microgram quantities of protein utilizing the prin ciple of protein-dye binding. Anal Biochem 72: 248-254, 1976. Caiozzo, VJ; MJ Baker; RE Herrick; M Tao; KM Baldwin. Effect of spaceflight on skeletal muscle: mechanical properties and myosin isoform content of a slow muscle. J Appl Physiol 76(4): 1764-1773, 1994. Callahan, LA; ZW She; TM Nosek. Superoxi de, hydroxyl radical, a nd hydrogen peroxide effects on single-diaphragm fi ber contractile apparatus. J Appl Physiol 90(1): 4554, 2001. Canon, F and F Goubel. Changes in stiffness induced by hindlimb suspension in rat soleus muscle. Pflugers Arch 429(3): 332-337, 1995.

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64 Capdevila, X; S Lopez; N Be rnard; E Rabischong; M Ra monatxo; G Martinazzo; C Prefaut. Effects of controlled mechani cal ventilation on respiratory muscle contractile properties in rabbits. Int Care Med 29: 103-110, 2003. Dean, R; F Shanlin; R Stocker; M Davies Biochemistry and pathology of radicalmediated protein oxidation. Biochem J 324: 1-18, 1997. Deschenes, MR; AA Britt; WC Chandler. A co mparison of the effects of unloading in young adult and aged skeletal muscle. Med Sci Sports Exerc 33(9): 1477-1483, 2001. Edgerton, VR; MY Zhou; Y Ohira; H Klitgaard; B Jiang; G Bell; B Harris; B Saltin; PD Gollnick; RR Roy. Human fiber size and en zymatic properties after 5 and 11 days of spaceflight. J Appl Physiol 78(5): 1733-1739, 1995. Esteban, A; I Alia; J Ibanez; S Benito; MJ T obin. Modes of mechanical ventilation and weaning. A national survey of Spanish hospitals. The Spanish lung failure collaborative group. Chest 106: 1188-1193, 1994. Furuno, K; MN Goodman; AL Goldberg. Role of different proteolytic systems in the degradation of muscle protei ns during denervation atrophy. J Biol Chem 265(15): 8550-8557, 1990. Grune, T and KJA Davies. Breakdown of oxidi zed proteins as a part of secondary antioxidant defenses in mammalian cells. Biofactors 6: 165-172, 1997. Grune, T; T Reinheckel; KJA Davies. Degrad ation of oxidized prot eins in K562 human hematopoietic cells by proteasome. J Biol Chem 271: 15504-15509, 1996. Grune, T; T Reinheckel; KJA Davies. Degrad ation of oxidized prot eins in mammalian cells. FASEB J 11: 526-534, 1997. Grune, T; T Reinheckel; M Joshi; KJA Davies. Proteolysis in culture d liver epithelial cells during oxidative stress. Role of the mulitcatalytic proteinase comples, proteasome J Biol Chem 270: 2344-2351, 1995. Haida, N; WM Fowler; RT Abresch. Effect of hindlimb suspension on young and adult skeletal muscle. Exp Neurol 103: 68-76, 1989. Kawakami, Y; H Akima; K Kubo; Y Mura oka; H Hasegawa; M Kouzaki; M Imai; Y Suzuki; A Gunji; H Kanehisa; T Fukunaga. Ch anges in muscle size, architecture, and neural activation after 20 days of bed re st with and without resistance exercise. Eur J Appl Physiol 84: 7-12, 2001. Klein, HHl S Piche; P Schuffe-Werner; P Niedmann; U Blattma nn; K Nebendahl. The effects of Trolox, a water-soluble vita min E analogue, in regionally ischemic, reperfused porcine hearts. Int J Cardiol 32: 291-302, 1991.

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65 Knisely, AS; SM Leal; DB Singer. Abnormaliti es of diaphragmatic muscle in neonates with ventilated lungs. J Pediatr 113(6): 1074-1077, 1988. Kondo, H; J Kodama; T Kishibe; Y Itokawa. Oxidative stress during recovery from muscle atrophy. FEBS 326: 189-191, 1993a. Kondo, H; I Nakagaki; S Sasaki; S Hori; Y Itokawa. Mechanism of oxidative stress in skeletal muscle atrophied by immobilization. Am J Physiol 265: E839-E844, 1993b. Lawler, JM and SK Powers. Oxidative stress, antioxidant status, and the contracting diaphragm. Can J Appl Physiol 23(1): 23-55, 1998. Le Bourdelles, G; N Viires; J Boczkowski; N Seta; D Pavlovic; M Aubier. Effects of mechanical ventilation on diaphragma tic contractile properties in rats. Am J Respir Crit Care Med 149: 1539-1544, 1994. Lowell, BB; NB Ruderman; MN Goodman. Regulation of myofibrillar protein degradation in rat skeletal muscle during brief and prolonged starvation. Metabolism 35 (12): 1121-1127, 1986. McDonald, KS and RH Fitts. Effect of hi ndlimb unloading on rat soleus fiber force, stiffness, and calcium sensitivity. J Appl Physiol 79(5): 1796-1802, 1995. Nagasawa, T; T Hatayama; Y Watanabe; M Tanaka; Y Niisato; DD Kitts. Free radicalmediated effects on skeletal muscle protein in rats treated with Fe-nitrilotriacetate. Biochem Biophys Res Comm 231 (1): 37-41, 1997. Powers, SK and K Hamilton. Antioxidants and exercise. Clinics in Sports Med 18(3): 525-536, 1999. Powers, SK; RA Shanely; JS Coombes; TJ Koesterer; M McKenzie; D Van Gammeren; M Cicale; SL Dodd. Mechanical ventilati on results in progressive contractile dysfunction in the diaphragm. J Appl Physiol 92 (5): 1851-1858, 2002. Radell, PJ; S Remahl; DG Nichols; LI Er iksson. Effects of prolonged mechanical ventilation and inactivity on piglet diaphragm function. Intensive Care Med 28: 358-364, 2002. Reid, M; K Haack; KM Franchek; PA Valberg; L Kobzik; MS West. Reactive oxygen in skeletal muscle: I. Intracellular oxidant kinetics and fatigue in vitro. J Appl Physiol 73(5): 1797-1805, 1992. Reid, M; DS Stokic; SM Koch; FA Khawli; AA Leis. N-acetylcysteine inhibits muscle fatigue in humans. J Clin Invest 94(6): 2468-2474, 1994.

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66 Reinheckel, T; N Sitte; O Ulrich; U Kuck elkorn; T Grune; KJA Davies. Comparative resistance of the 20S and 26S proteasome to oxidative stress. Biochem J 335: 637642, 1998. Riley, DA; GR Slocum; JL Bain; FR Sedlak ; TE Sowa; JW Mellender. Rat hindlimb unloading: soleus histochemistry, ultrastructure, and electromyography. J Appl Physiol 69(1): 58-66, 1990. Sassoon, CSH; VJ Caiozzo; A Manka; GC Sieck. Altered diaphragm contractile properties with controlled mechanical ventilation. J Appl Physiol 92: 2585-2595, 2002. Scott, JE; WM Cort; H Harley; DR Pa rrish; G Saucy. 6-hydroxychroman-2-carboxylic acids: novel antioxidants. J Am Oil Chem Soc 51: 200-203, 1974. Shanely, RA; JS Coombes; M Zergeroglu; AI Webb; SK Powers. Short-duration mechanical ventilation enhances diaphragma tic fatigue resistance but impairs force production. Chest 123: 195-201, 2003. Shanely, RA; MA Zergeroglu; SL Lennon; T S ugiura; T Yimlamai; D Enns; A Belcastro; SK Powers. Mechanical ventilation-indu ced diaphragmatic atrophy is associated with oxidative injury and in creased proteolytic activity. Am J Respir Crit Care Med 166(10): 1369-1374, 2002. Shindoh, A; A Dimarco; A Toma s; P Manubay; G Supinski. E ffect of N-acetylcysteine on diaphragm fatigue. J Appl Physiol 68(5): 2107-2113, 1990. Taillandier, D; E Aurousseau; D MeynialDenis; D Bechet; M Ferrara; P Cottins; A Ducastaing; X Bigard; C Guezennec; H Schm id; D Attaix. Coordi nate activation of lysosomal, Ca2+-activated and ATP-ubi quitin-dependent pr oteinases in the unweighted rat soleus muscle. Biochem J 316: 65-72, 1996. Usuki, F; A Yasukake; F Umehara; H Tokuna ga; M Matsumoto; K Eto; S Ishiura; I Higuchi. In vivo protection of a water-sol uble derivative of vitamin E, Trolox, against methylmercury-intoxication in the rat. Neuroscience Letters 304: 199-203, 2001. Van der Heijden; AB Kroese; PM Werker; MC de With; M de Smet; M Kon; P Bar. Improving the preservation of isolated rat skeletal muscles stored for 16 hours at 4 degrees C. Transplantation 69(7): 1310-1322, 2000. Waalkes, TP and S Undenfriend. A fluorometri c method for the estimation of tyrosine in plasma and tissues. J Lab Clin Med 50 (5): 733-736, 1957. Walker, MK; C Vergely; S lecour; C Ab adie; V Maupoil; L Rochette. Vitamin E analogues reduce the inciden ce of ventricular fibrilla tions and scavenge free radicals. Fundam Clin Pharmacol 12(2): 164-172, 1998.

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67 Wu, TW; N Hashimoto; JX Au; J Wu; DA Mickle; D Carey. Trolox protects rat hepatocytes against oxyradical damage and the ischemic rat liver from reperfusion injury. Hepatology 13(3): 575-580, 1991. Wu, TW; N Hashimoto; J Wu; D Carey; RK Li; DA Mickle; RD Weisel. The cytoprotective effect of Trolox w ith three types of human cells. Biochem Cell Biol 68(10): 1189-1194, 1990. Yang, L; J Luo; J Bourdon; M Lin; SB Gottf ried; BJ Petrof. Controlled mechanical ventilation leads to remodeling of the rat diaphragm. Am J Respir Crit Care Med 166: 1135-1140, 2002. Zeng, LH; J Wu; D Carey; TW Wu. Trolox a nd ascorbate: are they synergistic in protecting liver cells in vitro and in vivo? Biochem Cell Biol 69: 198-201, 1991. Zergeroglu, MA; MJ McKenzie; RA Shanely; D Van Gammeren; KC DeRuisseau; SK Powers. Mechanical ventilation-induced oxidative stress in the diaphragm. J Appl Physiol 95: 1116-1124, 2003.

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68 BIOGRAPHICAL SKETCH Jenna Betters was born in New Smyrna Beac h, Florida, and graduated salutatorian of her high school class in 1997. She received her bachelor’s degree in biology from the University of North Florida, Jacksonville, Florida, in May of 2001, where she was a member of Phi Kappa Phi Honor Society, University Scholar’s Honor Society, and Golden Key International Honor Society. Sh e began a master’s program in exercise physiology at the University of Florida, Ga inesville, Florida. She has worked for two years as a fitness trainer, and three years as a research assistant in the Molecular Physiology Laboratory within the Center for Exercise Science. Je nna has been accepted into the doctoral program and will pursue a Ph.D. in exercise physiology from the University of Florida.


Permanent Link: http://ufdc.ufl.edu/UFE0004290/00001

Material Information

Title: Trolox supplementation during mechanical ventilation attenuates contractile dysfunction and protein degradation
Physical Description: Mixed Material
Language: English
Creator: Betters, Jenna Leigh Jones ( Dissertant )
Criswell, David S. ( Thesis advisor )
Powers, Scott ( Reviewer )
Dodd, Steve ( Reviewer )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2004
Copyright Date: 2004

Notes

Abstract: Prolonged, controlled mechanical ventilation (MV) results in diaphragmatic atrophy and reduced diaphragmatic force generating ability. To investigate whether an antioxidant, Trolox, could attenuate atrophy and force loss, we tested the hypothesis that Trolox supplementation during MV would reduce protein degradation and contractile impairments of the diaphragm by preventing oxidative damage. Further, we postulated that proteolysis during MV is mediated by the ATP-ubiquitin-dependent proteasomal pathway. Sprague-Dawley rats were anesthetized, tracheostomized, and mechanically ventilated with 21% O₂ for 12 hours. Trolox was intravenously infused in a subset of ventilated animals. These were compared to groups of spontaneously breathing (SB) animals anesthetized for 12 hours, as well as an acutely anesthetized control group. Twelve hours of MV resulted in a 17% decrease in maximal tetanic force compared to controls. However, Trolox supplementation during MV completely attenuated the loss of maximal force. Proteolysis, measured as the release of free tyrosine from in vitro muscle strips, was increased 105% in MV animals compared to CON, but not different between CON and MV animals receiving Trolox. Lastly, the chymotrypsin-like activity of the 20S proteaseome was elevated in the MV animals (+76%), but Trolox attenuated this rise in activity. These data indicate that Trolox supplementation during MV completely attenuates MV-induced contractile dysfunction and proteolysis in the diaphragm.
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 78 pages.
General Note: Includes vita.
Thesis: Thesis (M.S.E.S.S.)--University of Florida, 2004.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

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Permanent Link: http://ufdc.ufl.edu/UFE0004290/00001

Material Information

Title: Trolox supplementation during mechanical ventilation attenuates contractile dysfunction and protein degradation
Physical Description: Mixed Material
Language: English
Creator: Betters, Jenna Leigh Jones ( Dissertant )
Criswell, David S. ( Thesis advisor )
Powers, Scott ( Reviewer )
Dodd, Steve ( Reviewer )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2004
Copyright Date: 2004

Notes

Abstract: Prolonged, controlled mechanical ventilation (MV) results in diaphragmatic atrophy and reduced diaphragmatic force generating ability. To investigate whether an antioxidant, Trolox, could attenuate atrophy and force loss, we tested the hypothesis that Trolox supplementation during MV would reduce protein degradation and contractile impairments of the diaphragm by preventing oxidative damage. Further, we postulated that proteolysis during MV is mediated by the ATP-ubiquitin-dependent proteasomal pathway. Sprague-Dawley rats were anesthetized, tracheostomized, and mechanically ventilated with 21% O₂ for 12 hours. Trolox was intravenously infused in a subset of ventilated animals. These were compared to groups of spontaneously breathing (SB) animals anesthetized for 12 hours, as well as an acutely anesthetized control group. Twelve hours of MV resulted in a 17% decrease in maximal tetanic force compared to controls. However, Trolox supplementation during MV completely attenuated the loss of maximal force. Proteolysis, measured as the release of free tyrosine from in vitro muscle strips, was increased 105% in MV animals compared to CON, but not different between CON and MV animals receiving Trolox. Lastly, the chymotrypsin-like activity of the 20S proteaseome was elevated in the MV animals (+76%), but Trolox attenuated this rise in activity. These data indicate that Trolox supplementation during MV completely attenuates MV-induced contractile dysfunction and proteolysis in the diaphragm.
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 78 pages.
General Note: Includes vita.
Thesis: Thesis (M.S.E.S.S.)--University of Florida, 2004.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0004290:00001


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TROLOX SUPPLEMENTATION DURING MECHANICAL VENTILATION
ATTENUATES CONTRACTILE DYSFUNCTION AND PROTEIN DEGRADATION














BY

JENNA LEIGH JONES BETTERS


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE IN EXERCISE AND SPORT SCIENCES


UNIVERSITY OF FLORIDA


2004

































Copyright 2004

by

Jenna Leigh Jones Betters

































This thesis is dedicated to my husband, Chad Betters, and my parents, Jim and Sue Jones,
for their love and support.















ACKNOWLEDGMENTS

This project would not be completed without the support and assistance of many

people. I would like to thank Dr. David Criswell, my committee chair and mentor, for his

time and assistance with this thesis. Also, Dr. Scott Powers and Dr. Steve Dodd served as

advisors to me during this process. I would especially like to thank Dr. Powers for the use

of his laboratory equipment to complete this study.

Dr. R. Andrew Shanely, Darin van Gammeren, Darin Falk, and Dr. Keith

DeRuisseau devoted their time and talents to completing this project. I thank them for all

of the early mornings, as well as the late nights. I also thank Tossaporn Yimlamai for his

assistance with measuring the proteasome activity.

Lastly, I thank my husband, Chad Betters, for helping me through the tough

moments, and my parents, Jim and Sue Jones, for encouraging me to persevere through

challenges.
















TABLE OF CONTENTS
page

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

L IST O F TA B LE S ..................... ............. ................................... .. ................ .. vii

LIST OF FIGU RE S ........................................ ............ .............. .. viii

ABSTRACT ........ .............. ............. ...... ...................... ix

CHAPTER

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

B a ck g ro u n d .................................................................................................... ..... .
Significance of the Study ........................................................... .............6

2 L IT E R A TU R E R E V IE W .................................................................. .....................8

Skeletal M uscle A daptations to U nloading ........................................ .....................8
Oxidative Stress and Skeletal Muscle............................................ 19
Skeletal Muscle Unloading and Protein Degradation.....................................21
Antioxidant Supplementation and Skeletal Muscle.................................................23
Sum m ary ...................................... ................. ................. .......... 27

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

E x p erim mental D design ......................................................................... ................... 2 8
D iaphragm Contractile Function ........................................ .......................... 30
Protein D degradation .................. ................................ ...... .. .......... .... 32
20S Proteasom e A activity .................................................. .............................. 33
Total and N on-protein Thiols .............................................................................. 33
Statistical A nalysis................................................... 35

4 R E S U L T S .............................................................................3 6

Systemic and Biologic Responses to Treatment..................................................36
Effects of Anesthesia on Diaphragm Contractile Properties ................. ............36
Effects of Mechanical Ventilation on Contractile Properties.................................36
Effects of Trolox on Contractile Properties..................................... ............... 37
Protein D degradation .................. ..................................... .. ............ 38









20S Proteasom e A activity ................................................................. ............... 38
O x id ativ e Stress ........ ......................................................................... 3 8

5 D IS C U S S IO N ...............................................................................................4 8

Trolox Attenuates Mechanical Ventilation-induced Contractile Dysfunction and
Proteolysis in the Rat Diaphragm:Introduction .................................................48
M materials and M methods ....................................................................... ..................49
R esu lts .............. .... ..... ............. ... ............................................55
D discussion ............. ..... .. ..................................... .......... 57

LIST O F R EFEREN CE S ......... .......................................................... ............... 63

B IO G R A PH IC A L SK E TCH ..................................................................... ..................68
















LIST OF TABLES


Table p

4-1 Body and Diaphragm Weights of Control, Spontaneously Breathing, and
M mechanically Ventilated Animals.......... .. ........ ................................... 39

4-2 Maximal Isometric Twitch and Tetanic Force of Control, Spontaneously
Breathing, and Mechanically Ventilated Animals ........................ .................39

4-3 Contractile Parameters of Maximal Isometric Twitch and Tetanic Forces of
Control, Spontaneously Breathing, and Mechanically Ventilated Animals.............40
















LIST OF FIGURES


Figure page

4-1 Force-frequency responses. .............................................. ............... 41

4-2 Responses of in vitro diaphragm strips to a 30-min fatigue protocol. ..................42

4-3 Percent of initial force maintained by in vitro diaphragm strips after a 30-min
fatigue protocol. .....................................................43

4-4 Total in vitro diaphragm 4ic protein degradation as measured by the rate of
tyrosine release during a 2-hour incubation. ................ .................. .............44

4-5 Chymotrypsin-like activity of the 20 S proteasome in diaphragm tissue..............45

4-6 Total thiol concentration in diaphragm tissue. ............. .................. .........46

4-7 Non-protein thiol concentration in diaphragm tissue ....................................47


....................... ...............47















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science in Exercise and Sport Sciences

TROLOX SUPPLEMENTATION DURING MECHANICAL VENTILATION
ATTENUATES CONTRACTILE DYSFUNCTION AND PROTEIN
DEGRADATION


By

Jenna Leigh Jones Betters

May 2004

Chair: David Criswell
Major Department: Exercise and Sport Sciences


Prolonged, controlled mechanical ventilation (MV) results in diaphragmatic

atrophy and reduced diaphragmatic force generating ability. To investigate whether an

antioxidant, Trolox, could attenuate atrophy and force loss, we tested the hypothesis that

Trolox supplementation during MV would reduce protein degradation and contractile

impairments of the diaphragm by preventing oxidative damage. Further, we postulated

that proteolysis during MV is mediated by the ATP-ubiquitin-dependent proteasomal

pathway. Sprague-Dawley rats were anesthetized, tracheostomized, and mechanically

ventilated with 21% 02 for 12 hours. Trolox was intravenously infused in a subset of

ventilated animals. These were compared to groups of spontaneously breathing (SB)

animals anesthetized for 12 hours, as well as an acutely anesthetized control group.

Twelve hours of MV resulted in a 17% decrease in maximal tetanic force compared to









controls. However, Trolox supplementation during MV completely attenuated the loss of

maximal force. Proteolysis, measured as the release of free tyrosine from in vitro muscle

strips, was increased 105% in MV animals compared to CON, but not different between

CON and MV animals receiving Trolox. Lastly, the chymotrypsin-like activity of the 20S

proteaseome was elevated in the MV animals (+76%), but Trolox attenuated this rise in

activity. These data indicate that Trolox supplementation during MV completely

attenuates MV-induced contractile dysfunction and proteolysis in the diaphragm.














CHAPTER 1
INTRODUCTION

Weaning patients from a mechanical ventilator is a serious clinical issue.

Mechanical ventilation (MV) is characteristically used to maintain alveolar ventilation in

patients who are incapable of ventilation on their own. As such, MV is an important life-

preserving measure. However, removing patients from the ventilator, also known as

weaning, can be difficult in many cases. Weaning procedures account for more than 40%

of total MV time in patients who have difficulty weaning from the ventilator (Esteban

1994), suggesting that this is a serious clinical issue. Diaphragmatic weakness, the result

of atrophy, is a major cause of difficult weaning. Therefore, the mechanisms underlying

the rapid loss of diaphragm mass and strength during periods of MV should be explored.

Background

In many clinical situations, patients are unable to maintain adequate alveolar

ventilation. In these cases, MV is necessary for life support. This may occur during acute

respiratory failure, surgeries involving general anesthesia, diseases such as sepsis, and

with pre-term infants whose lungs and respiratory muscles are not completely developed.

Unfortunately, removing a person from MV is not always simple since even short periods

of MV can weaken the diaphragm to the point where resumption of normal loading leads

to diaphragm fatigue and respiratory failure. The process of gradually weaning patients

from a ventilator can result in extended hospital stays, as well as additional costs to the

patients, insurance companies, and hospitals.









The mechanisms) behind MV-induced diaphragmatic weakness are unclear at this

time. However, recent research by Powers and colleagues (2002) has reported that MV-

induced diaphragmatic dysfunction is intrinsic to the muscle and increases in magnitude

with increasing time on the ventilator. Oxidative stress is one potential mediator of MV-

induced diaphragmatic dysfunction. Oxidative stress is the result of an imbalance

between reactive oxygen species (ROS) production and antioxidant protection (Lawler

and Powers 1998), and has been implicated as a contributor in numerous pathological

conditions, including atherosclerosis, obstructive lung disease, aging, and fatigue of

skeletal muscle. Although ROS are continuously produced in human beings, a balance is

generally maintained between ROS production and cellular antioxidant systems.

However, periods of stress, whether from trauma, ischemia, infection, etc., lead to an

increase in the formation of ROS, which may overwhelm antioxidant systems causing

oxidative stress. This oxidative stress can cause lipid peroxidation, damage to DNA and

proteins, and cell death.

It is known that critical illnesses like sepsis or adult respiratory distress syndrome

can drastically increase the ROS production and lead to oxidative stress in skeletal

muscle. This is significant because oxidized proteins are more prone to proteolytic attack

and degradation (Grune et al. 1995, Grune et al. 1996, Dean et al. 1997, Nagasawa et al.

1997). A similar mechanism may contribute to protein loss in the diaphragm during MV.

During periods of muscle disuse, ROS production has been shown to increase (Kondo et

al. 1993a, Kondo et al. 1993b). Further, new data indicate significant increases in lipid

peroxidation and protein oxidation in the diaphragms of mechanically ventilated rats

(Shanely et al. 2002, Zergeroglu et al. 2003), and a corresponding increase in total in









vitro protein degradation (Shanely et al. 2002). Therefore, it seems logical that

diaphragmatic weakening during MV-induced unloading may be caused by oxidative

damage to contractile proteins leading to heightened proteolytic degradation.

Problem Statement

Since prolonged controlled MV results in a significant loss of diaphragmatic

maximal force production (LeBourdelles et al. 1994, Powers et al. 2002, Radell et al.

2002, Sassoon et al. 2002, Yang et al. 2002, Capdevila et al. 2003, Shanely et al. 2003),

and is associated with evidence of increased oxidative stress (Powers et al. 2002, Shanely

et al. 2002, Zergeroglu et al. 2003), we postulate a causal relationship and will seek to

examine the efficacy of antioxidant infusion during MV for the preservation of

diaphragm function. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a

water-soluble vitamin E analog, is an effective scavenger for a variety of radicals (Walker

et al. 1998). This antioxidant prolongs survival of many cell types exposed to oxyradicals

(Wu et al. 1991). Therefore, we will specifically determine whether Trolox

supplementation during 12 hours of controlled mechanical ventilation will attenuate MV-

induced diaphragmatic contractile dysfunction, oxidative stress, and protein degradation.

Variables in Study

Independent variables. We will manipulate mechanical ventilation and Trolox

supplementation.

Dependent variables. We will measure diaphragm contractility, tyrosine release

as a measure of degradation, 20S proteasome activity, and markers of oxidative stress

such as protein carbonyls.









Control variable. We will only study female Sprague-Dawley rats, so gender is

purposely excluded from this study. The animals will be young adult rats (~4 months

old), thus maturation and aging effects are excluded from the study.

Extraneous variable. We will not control P02 levels in these animals, so hypoxic

conditions will not be controlled. Pilot experiments have been conducted to confirm that

the MV protocol maintains normal arterial P02 and PC02 levels. However, the

spontaneous breathing animals are expected to be mildly hypoxic and hypercapnic due to

the effects of the anesthesia. To assess the potential effects of these conditions on

diaphragm function, a separate group of rats will be studied without exposure to MV or

spontaneous breathing protocols (pure controls).

Hypotheses

We hypothesize that:

1.) Twelve hours of controlled MV will induce contractile dysfunction in the rat
diaphragm compared to controls.

2.) Twelve hours of controlled MV will increase oxidative stress levels in the diaphragm
muscle compared to control diaphragms.

3.) Twelve hours of controlled MV will increase the rate of total protein degradation
within the diaphragm compared to control diaphragms.

4.) Infusion of Trolox during 12 hours of MV will attenuate the diaphragmatic
dysfunction, reduce the rate of protein degradation, and decrease oxidative stress levels
compared to controls.


Definition of Terms

Controlled mechanical ventilation (MV). Tracheostomized animals will receive

all breaths from the volume-controlled small-animal ventilator (Harvard Apparatus). The

tidal volume will be ~1 ml/100 g body weight with a respiratory rate of 80 breaths/min.









Positive end-expiratory pressure of 1 cm H20 will be used throughout the protocol.

Therefore, the diaphragm muscle will be effectively unloaded.

Spontaneous breathers (SB). Animals receiving sham surgeries and 12 h of

anesthesia, without controlled MV.

Antioxidant. A compound capable of preventing or delaying damage from

oxidative stress.

Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-

soluble vitamin E analog with antioxidant characteristics.

Reactive oxygen species (ROS). Molecules derived from molecular oxygen that

have an unpaired electron in their outer orbital, making them highly reactive.

Oxidative stress. An imbalance between a greater production of reactive oxygen

species and reduced antioxidant protection.

Proteolysis. The process whereby proteins are broken down into peptide

fragments and amino acids. This occurs through three main pathways: (1) lysosomal

proteases (cathepsins), (2) Ca2+-dependent cysteine proteases (calpains), and (3) the ATP-

dependent, ubiquitin-proteasome pathway.

Ubiquitin. A protein found in all cell types that acts as a molecular tag when

attached to proteins, marking them for degradation by the proteasome.

Tyrosine. An amino acid that is neither synthesized nor degraded in skeletal

muscle. The net accumulation of this amino acid, assayed fluorometrically within the

incubation buffer, reflects net in vitro protein degradation within muscle.









Limitations/Delimitations/Assumptions

Limitations. The invasive nature of this research negates the use of human

subjects. A rat model has been chosen to study the diaphragm muscle because of the

similarities in structure and function between the rat diaphragm and human diaphragm.

Trolox does not readily dissolve in saline. Addition of sodium hydroxide (NaOH)

was necessary to solubilze Trolox. This increased the pH of the Trolox solution well

above the physiological range. As a result, the spontaneous breathing group receiving

Trolox was limited to only 3 animals that survived the 12-h protocol. These animals were

not included in statistical analyses, thus we were limited in our ability to control for

Trolox infusion without MV.

Delimitations. Gender and species differences may exist in regard to the efficacy

of Trolox as a protectant against diaphragmatic dysfunction. We have chosen to study

only female Sprague-Dawley rats.

Assumptions. It is assumed that the diaphragm is completely unloaded during

MV. Previous experiments have inserted electromyographic (EMG) needles into the

muscle and found that it is silent during controlled MV (Le Bourdelles et al. 1994,

Powers et al. 2002).

Significance of the Study

Atrophy and protein degradation occurring within an unloaded diaphragm muscle

result in an 18% reduction in force generation with just 12 hours of MV (Powers et al.

2002). This force loss increases to 46% with 24 hours of MV. Eighteen hours of MV is

associated with a significant increase in lipid peroxidation and protein oxidation (Shanely

et al. 2002). This research will improve our knowledge of the mechanisms associated

with MV-induced diaphragmatic dysfunction. It will provide insight into clinical






7


strategies using antioxidants to attenuate diaphragmatic atrophy incurred during MV so

that patients may be removed more swiftly and successfully from the ventilator.














CHAPTER 2
LITERATURE REVIEW

The diaphragm is an essential muscle for the maintenance of normal ventilation in

mammals. This muscle has an activity level greater than most other skeletal muscles,

which puts it at increased risk for atrophy and dysfunction during prolonged periods of

inactivity such as mechanical ventilation (MV). The purpose of this study is to determine

whether Trolox supplementation during MV will attenuate MV-induced diaphragmatic

dysfunction related to oxidative stress and protein degradation. This chapter provides a

critical review of the scientific literature related to the proposed project. All pertinent

articles in the specified areas will be covered. In some cases, however, the literature

abounds and only a few representative articles will be reviewed in detail with cursory

reference to other corroborating evidence. Whenever possible, interpretations of the

reviewed data will be offered based on perceived consensus in the literature.

This review is organized under the following headings: (a) Skeletal muscle

adaptations to unloading, (b) Oxidative stress and skeletal muscle, (c) Skeletal muscle

unloading and protein degradation, and (d) Antioxidant supplementation and skeletal

muscle.

Skeletal Muscle Adaptations to Unloading

Muscle atrophy, the result of disuse, develops rapidly after immobilization (Appell

et al. 1997). Several models of disuse exist that either prevent the loading of skeletal

muscle with normal body weight, or eliminate the effect of gravity on the upright

position. These models include hindlimb unloading in rats, casting of limbs in animals









and humans, spaceflight or simulation of a microgravity environment, bed rest, and

denervation.

Atrophy and Fiber Type Shifts with Unloading

Fast and slow twitch locomotor muscles undergo considerable atrophy with

unloading (McDonald and Fitts 1995). Simultaneously, unloaded locomotor muscles

exhibit a fiber type conversion from type I to type II fibers (Haida et al. 1989). The

greatest change occurs in antigravity, slow twitch muscles such as the soleus. After 1, 2,

and 3 wk of hindlimb unloading (HU) in Sprague-Dawley rats, mean mass of the soleus

was decreased by 28, 44, and 56%, respectively (McDonald and Fitts 1995). Mean fiber

diameter decreased with increasing length of HU. Riley et al. (1990) demonstrated that

14 days of unloading caused a reduction in types I and IIa cross-sectional areas (CSA) of

63 and 47%, respectively. They also showed that HU reduced the muscle-to-body weight

ratio showing muscle-specific effects of the unloading treatment.

A decrease in the percentage of slow twitch fibers, with an increase in the

percentage of fast twitch fibers, leads to an increase in maximal shortening velocity

(Canon and Goubel 1995). A 4 wk hindlimb suspension study led to significant fiber

atrophy in both the soleus and extensor digitorium longus (EDL) muscles (Deschenes et

al. 2001). A significant decrease in the percentage of type I fibers was noted in unloaded

solei. This was accompanied by an increase in the percentages of types IIa and IIx/b

fibers. The loss of fiber size is of concern because force production is directly related to

fiber size. Muscle unloading failed to induce significant fiber type conversion within the

fast twitch EDL muscle.

Like hindlimb unloading, casting, or limb fixation, results in muscle atrophy

(Booth 1977, Kondo et al. 1993a, Kondo et al. 1993b, Appell et al. 1997). Both the onset










and the degree of atrophy of limb muscles during casting immobilization are dependent

on the length of the muscle during limb fixation (Booth 1977). Booth (1977) performed

two sets of experiments using a rat model, one with the ankle and foot fixed in slight

plantar flexion (PF), and one with the foot fixed in dorsal flexion (DF). In PF, the calf

muscles are slightly less than resting length, while they are slightly lengthened in DF.

Weight losses of the gastrocnemius, soleus, quadriceps, and white portion of the vastus

lateralis were exponential between days 2-10 of PF immobilization. During 28 days of PF

immobilization, the gastrocnemius muscle atrophied by 51%, and the plantaris atrophied

by 48%. In contrast, there was no change in the weight of the stretched tibialis anterior

muscle during PF immobilization. When the ankle was casted in DF, the calf muscles

were stretched beyond resting length and a delay in the onset of atrophy was noted.

Appell et al. (1997) reported similar findings. Eight days of casting in male Wistar rats

with the soleus muscle in a shortened position lead to a 35% atrophy compared to control

muscles. These data clearly demonstrate that a greater degree of atrophy, as well as an

earlier onset of atrophy, is seen in muscles fixed in positions that are less than resting

length.

Seven days of casting the ankle joint of one hindlimb in the fully extended position

resulted in a loss of -45% of soleus muscle weight (Kondo et al. 1993a). In a similar

experiment, 4-, 8-, and 12-day immobilization of one hindlimb by casting led to

decreases of 49, 60, and 81% of control soleus muscle weight, respectively (Kondo et al.

1993b). The activity of xanthine oxidase (XOD) increased significantly in the atrophied

muscles. Type O (superoxide-producing) XOD was -2.3 times higher in immobilized

muscle than in control muscle. Also, the substrates of XOD, xanthine and hypoxanthine,










increased in atrophy. Therefore, superoxide-generating XOD may be more active in the

atrophied muscle, meaning oxidative stress is accompanying atrophy. These observations

formed the basis of our original hypothesis that oxidative stress may be associated with

diaphragmatic atrophy.

Adaptations in the size, metabolic properties, and vascularity of muscle fibers occur

in space where the gravity-dependent load of the body on muscles is absent (Edgerton et

al. 1995). Vastus lateralis muscle fibers sampled from astronauts before and after

spaceflights showed postflight biopsies with 6-8% fewer type I fibers than preflight. This

loss of type I fibers seemed to be accounted for by an increase in type IIa fibers. Mean

fiber CSAs were 16-36% smaller after the flight. Little difference in percent atrophy was

found in type I versus type II fibers. The number of capillaries per fiber was 24% lower

after flight as compared to before flight. Spaceflight resulted in an increase in the

myofibrillar ATPase activity of type II fibers, whereas alpha-glycerophosphate

dehydrogenase (GPD) activity was 80% higher in type I fibers after flight. This study

found significant variability between subjects that was attributed to the volume and kind

of physical work that each astronaut performed during flight. The results indicate that the

degree of atrophy may have been related to the type of physical activities undertaken

during spaceflight.

Contractile Dysfunction with Unloading

Maximal isometric specific tension (Po) of the soleus was significantly reduced

after 1 and 2 wk of hindlimb suspension compared to normal loaded solei in Sprague-

Dawley rats (McDonald and Fitts 1995). A reduction in the number of cross bridges per

fiber area, and possibly a reduced force per cross bridge, may explain the decrease in Po.










The gravity-dependent load of the human body in the upright position is essential

for maintenance of lower limb skeletal muscle function (Berg et al. 1997). In particular,

contractile properties of slow, antigravity skeletal muscle are sensitive to the

microgravity environment. Six days of spaceflight induced contractile changes in the

soleus muscles of male Sprague-Dawley rats (Caiozzo et al. 1994). The force-velocity

relationship, force-frequency relationship, and fatigability were studied in situ 3 h after

landing. Maximal isometric tension (Po) was decreased by 24% and maximal shortening

velocity was increased by 14% in flight muscles. The flight muscles's force-frequency

curve was shifted to the right of the control muscles's curve. Control muscles generated

64% of the initial Po after the fatigue protocol, while flight muscles only generated 36%

of initial Po.

Bed rest results in similar changes in skeletal muscle function. After 6 wk of bed

rest in 7 healthy men, maximum voluntary isometric and concentric knee extensor torque

decreased across angular velocities by 25-30% (Berg et al. 1997). Type I fiber cross-

sectional area (CSA) of the vastus lateralis decreased by 18.2%. No change in CSA or

fiber diameter was apparent in either type IIa or lib fibers. The greater loss in strength

compared to muscle CSA suggests specific tension of muscle and/or neural input to

muscle is reduced. Another study showed that 20 days of bed rest decreased maximal

knee extension force by 10.9% (Kawakami et al. 2001). The reduction of muscle strength

in this study was likely due to a decreased ability to activate motor units.

In conclusion, antigravity, slow twitch muscles such as the soleus are more

susceptible to changes as a result of unloading than are nonpostural, phasic muscles such

as the EDL. These changes include fiber atrophy, reduced force production, and a









conversion to a faster type muscle. A decreased number of active cross bridges per

volume of muscle after unloading may explain the loss in specific tension seen in humans

and animals.

Mechanical Ventilation and Diaphragm Atrophy

An early epidemiological study found that some infants and neonates who had

received long-term ventilatory assistance (>12 days) had subnormal diaphragmatic

muscle mass on gross necropsy examination (Knisely et al. 1988). The retrospective

study examined sections of the costal diaphragm, along with portions of the infrahyoid

strap muscle and the posterior portion of the tongue. These extra-diaphragmatic sites

appear to be coordinated with diaphragmatic function in infants. Histologic findings in

the diaphragms of neonates and infants supported by MV for at least 12 days were

consistent with disuse atrophy, denervation atrophy, or failure of normal growth and

maturation. Myofibers from the other two sites appeared normal. The researchers

concluded that long-term ventilatory assistance predisposes diaphragmatic myofibers to

disuse atrophy or to failure of normal growth. This weakening may play a role in difficult

weaning procedures from ventilatory support. Thus, mechanical ventilation unloads the

diaphragm muscle like hindlimb suspension, casting, spaceflight, bed rest, and

denervation unload other major skeletal muscles.

Several studies have shown that controlled MV leads to diaphragmatic atrophy (Le

Bourdelles et al. 1994, Shanely et al. 2002, Yang et al. 2002, Capdevila et al. 2003) and

reductions in protein content (Shanely et al. 2002). Shanely et al. (2002) observed a

significant decrease in both total and costal diaphragm masses after 18 h of MV in rats,

but no losses of body mass or soleus mass. All four diaphragmatic myosin heavy chain

(MHC) types experienced a reduction in CSA. However, type IIx and lib fibers atrophied









to a greater extent than type I fibers. This contrasts with results seen in locomotor muscle

during periods of atrophy, where type I fibers typically atrophy more than type II fibers.

In rats mechanically ventilated for up to 4 days, a significant decrease in diaphragm

weight / body weight was seen, which amounted to a mean reduction of 13.4% compared

to controls (Yang et al. 2002). These researchers noted a shift in myosin heavy chain

(MHC) isoform from slow-to-fast. There was a decrease in the percentage of fibers

expressing type I MHC, while the number of fibers co-expressing both type I and type II

MHC increased in the diaphragm (12.5% vs. 3% in controls). In contrast, the percentages

of type I, type II, and hybrid fibers remained unchanged in the limb muscles after MV.

The combination of mechanical unloading, reduced electrical activity, and intermittent

passive shortening in the diaphragm during MV may be powerful stimuli for MHC

transformations. These modifications may alter the maximal specific force and fatigue

resistance of the diaphragm following MV.

In contrast, Sassoon et al. (2002) found that 3 d of MV did not alter fiber type

proportions or their relative contribution to total CSA in a rabbit model. Like Sassoon et

al. (2002), Capdevila and colleagues (2003) found no significant alterations in fiber type

proportions following 51+3 h in the rabbit diaphragm. It is possible that species

differences contributed to the discrepancy between these studies and Yang et al. (2002)

since fiber type composition differs in rat and rabbit diaphragms.

Mechanical Ventilation-induced Contractile Dysfunction

Eight studies have been published which confirm that controlled mechanical

ventilation alters diaphragm contractile properties. Although different animal models

were employed, all of these studies agree that MV significantly reduces diaphragmatic

force-generating capacity. One of the earliest studies, performed by Le Bourdelles et al.












(1994), found that 48 h of MV in rats significantly decreased in vitro diaphragm

contractility compared to controls, while the soleus and EDL muscles's contractility were

unaffected. No electrical activity of the diaphragm was detected during MV in the 2

animals in which it was measured. Diaphragmatic force-generating capacity was reduced

by 41.5% compared to spontaneously breathing controls. The data did not show a

difference in total protein content, or citrate synthase or lactate dehydrogenase enzyme

activities between control and MV diaphragms. Their data indicate that the decreased

force generation did not result from decreased muscle mass as typically seen with general

disuse atrophy. An important finding from this study was that the level of sodium

pentobarbital required to maintain a surgical plane of anesthesia over a 2-day period did

not induce locomotor muscle atrophy, nor did it impair locomotor muscle maximal

tetanic force generation. This is significant because it demonstrates that MV itself exerts

deleterious effects on diaphragmatic function independently of anesthesia.

Anzueto et al. (1997) mechanically ventilated adult baboons (n=7) for 11 days and

showed that maximum transdiaphragmatic pressure decreased by 25% from day 0 to day

11. In addition, diaphragmatic endurance decreased by 36% from day 0 to day 11. These

animals were infused with a long-acting neuromuscular blockade, pancuronium (10

jag/kg/h), which may have contributed to the results. However, pancuronium was

withheld on day 11, and reversal of neuromuscular blockade was noted. No major

changes occurred in hemodynamics, oxygenation, or lung function. Arterial pressure,

pulmonary artery pressure, pulmonary artery occlusion pressure, and cardiac output

remained constant over the 11-day period. The absolute force-frequency curves showed a










decrease in diaphragmatic response to all frequencies of stimulation tested. Prolonged

MV in this baboon model resulted in impaired diaphragmatic strength and endurance.

Five days of volume-controlled MV in a piglet model resulted in depressed

diaphragm contractility and activation (Radell et al. 2002). However, nerve conduction

and transmission were unaffected. Bipolar transvenous pacing catheters were used to

stimulate the phrenic nerve and pace diaphragm contractions during measurements. The

researchers found that transdiaphragmatic pressure (Pdi) decreased over time at all

frequencies tested. By day 5, the drop in Pdi was greater than 20% at all frequencies.

There was a 30% decrease in compound muscle action potential (CMAP) amplitude of

the costal diaphragm from day 1 to day 5. The stable response to repetitive stimulation

does not support neuromuscular transmission failure as the cause of dysfunction. Instead,

the decrease in CMAP amplitude and fall in force output are indicative of excitation-

contraction (E-C) coupling or membrane depolarization as mechanisms leading to

diaphragmatic dysfunction. Thus, this study found that nerve conduction and

neuromuscular transmission are unaffected during prolonged MV, but the diaphragm

does experience a loss in function that may originate at the level of the muscle cell

membrane or the contractile apparatus.

Yang et al. (2002) also found that maximal twitch and tetanic force generating

capacity were significantly lowered in rats mechanically ventilated for up to 4 days as

compared to controls. There were no significant differences in contraction time, half-

relaxation time, or fatigue resistance. Optimal muscle length, Lo, was found to be shorter

in the MV group compared to controls and anesthetized spontaneous breathers. This may

provide indirect evidence for a loss of sarcomeres in series.










A study by Powers et al. (2002) examined the time course of MV-induced

diaphragmatic contractile dysfunction in an in vitro diaphragm strip preparation. When

compared to control rats, MV of 12, 18, and 24 h duration resulted in a right shift in the

force-frequency curve of the diaphragm. The magnitude of the curve's right shift was

dependent upon the duration of MV. Twelve h of MV resulted in an 18% reduction in the

mean diaphragmatic specific tension, while 24 h of MV resulted in a 46% reduction. This

experiment also included two groups of spontaneously breathing (SB) animals that were

maintained on a surgical plane of anesthesia for 18 and 24 h. Analysis of arterial blood

gas tensions and pH revealed that these groups experienced hypoxemia, hypercapnia, and

mild acidosis. These disturbances were likely due to hypoventilation resulting from

depressed ventilatory drive. Nevertheless, like Le Bourdelles and coworkers (1994), this

lab found that the level of pentobarbital sodium required to maintain a surgical plane of

anesthesia did not impair in vitro diaphragmatic function in the spontaneously breathing

animals. The researchers concluded that MV-induced contractile dysfunction was due to

intrinsic changes within diaphragm fibers. These may include a reduction in the

myofibrillar protein concentration, abnormalities of contractile or cytoskeletal proteins,

and/or impaired excitation-contraction (E-C) coupling.

Controlled MV also had a time-dependent deleterious effect on diaphragm

contractility in a rabbit model (Sassoon et al. 2002). Transdiaphragmatic pressure

decreased to 63% of controls after 1 day of MV, and to 49% after 3 days. Similarly, in

vitro tetanic force decreased to 86% of control values after 1 day, and to just 44% of

controls after 3 days. A major finding of this study was that diaphragm muscle injury

accounted for 66% of the variance in the reduction of tetanic force. Significant myofibril










damage was found in diaphragms after 3 days of MV, but not in soleus muscles from the

same animals. There was no myofibril damage in control diaphragms. Another study in

rabbits mechanically ventilated for 49 1 h also found evidence of altered diaphragm

fiber ultrastructure indicative of fiber injury in the MV group (Bernard 2003). Disruption

and fragmentation of myofibrils were observed in diaphragms after MV, along with an

increase in the size of the interfibrillar space and in the size and number of sarcoplasmic

lipid vacuoles. The mechanism for myofibril injury with inactivity is unknown, but may

contribute to MV-induced diaphragmatic dysfunction and difficulties in weaning patients

from ventilators.

The laboratory of Capdevila et al. (2003) examined both the diaphragm and 5th

external intercostal muscle following 51+3 h of MV in rabbits. MV significantly

decreased Po compared to controls by 25%. This was significantly worsened after a

fatigue run. Diaphragmatic and 5th external intercostal muscle masses were significantly

reduced following MV. The MV rabbits had lower peak tetanic tensions, reduced fatigue

resistance indices, and increased relaxation times compared to control diaphragms. The

force reduction of the diaphragm was most likely related to the change in mass.

Eighteen h of controlled MV in a rat model significantly reduced both

diaphragmatic maximal twitch force production and Po (-20%) compared to controls

(Shanely et al. 2003). However, diaphragms from MV-treated animals maintained a

significantly greater fatigue resistance compared to control animals. That is, MV

diaphragms maintained higher relative forces throughout a 30-min fatigue test. When

absolute force production was compared, however, the control diaphragms produced

higher specific forces than MV diaphragms during the fatigue test. Interestingly, costal










diaphragm citrate synthase, total superoxide dismutase (SOD), Cu-Zn-SOD, and Mn-

SOD activities were significantly greater in the MV animals than the control animals.

These findings indicate that 18 h of MV improves diaphragmatic fatigue resistance

relative to maximal force. However, long duration MV (weeks to months) does impair

diaphragmatic endurance.

Oxidative Stress and Skeletal Muscle

Reactive oxygen species (ROS) production results from a number of biochemical

reactions, most notably aerobic metabolism (Lawler and Powers 1998). Infection,

inflammation, strenuous exercise, and obstructive lung disease are a few conditions that

increase diaphragm exposure to ROS.

Skeletal Muscle Atrophy and Dysfunction Related to Oxidative Stress

Kondo et al. (1993a) have found that muscle atrophy induced by immobilization is

accompanied by oxidative stress. Thiobarbituric acid reactive substance (TBARS) and

oxidized glutathione (GSSG) were increased, while total glutathione (GSH) was reduced

in atrophied soleus muscle from immobilized hindlimbs. The ankle of one hindlimb of

male Wistar rats was immobilized with the soleus muscle in a shortened position. Some

rats were sacrificed after 7 days of immobilization (Atrophy group), while the ankle

joints of other rats were remobilized for another 5 days (Recovery group). The TBARS

level in atrophic muscle increased significantly in the Recovery group, indicating a rise in

lipid peroxidation. GSSG levels were significantly greater in atrophic muscle than that in

contralateral control muscle. Total GSH level decreased significantly in atrophic muscle.

Both the increase of TBARS and GSSG imply enhanced oxidative stress during muscle

recovery from atrophy.












The diaphragm muscle is susceptible to alterations in contractility resulting from a

direct effect of hydroxyl radical (OH ) and superoxide anion radical (02 '-) on

contractile proteins (Callahan et al. 2001). Chemically skinned (Triton X-100) single rat

diaphragm fibers exposed to 02 had a significant 14.5% reduction in maximum

calcium-activated force. Exposure to OH significantly decreased maximum calcium-

activated force by 43.9%. Hydrogen peroxide (H202) did not affect maximum force or

calcium sensitivity. The effects of OH and 02 on contractility may contribute to the

characteristic respiratory muscle dysfunction seen in certain pathophysiological

conditions such as sepsis and skeletal muscle fatigue.

When proteins are damaged by ROS, their function is impaired, and their

susceptibility to proteolysis is enhanced (Nagasawa et al. 1997). Oxidatively modified

proteins are easily degraded by the proteasome, a multisubunit proteinase. Rats given an

intraperitoneal injection of ferric nitrilotriacetate (FeNTA) and sacrificed at 1.5, 3, and 6

h after injection had significant modification of muscle proteins after an iron overload

(Nagasawa et al. 1997). Protein carbonyl content of both soleus and EDL muscles was

elevated up to 3 h after injection. These results show that muscle proteins were modified

by free radicals generated from the FeNTA injection. The rate of tyrosine release reached

a maximum at 3 h after injection, suggesting an increase in the rate of total protein

degradation up to 3 h after the onset of an oxidative stress. Myosin and actin responded

strongly to specific antibody against 2,4-dinitrophenyl group, implying that these

myofibrillar proteins were drastically modified by free radicals. These results suggest that

oxidatively modified muscle proteins undergo rapid proteolysis.









MV-induced Oxidative Stress

Shanely et al. (2002) found that oxidative stress, specifically protein carbonyl

content and total 8-isoprostane concentration, was increased after 18 h of MV in rats.

Protein carbonyl levels significantly increased by 44%, while 8-isoprostane concentration

increased 53% compared with controls. Short-term controlled MV of just 6 h was

sufficient to increase oxidative injury in the diaphragm of rats (Zergeroglu et al. 2003).

Reactive protein carbonyl derivatives (RCD) and lipid hydroperoxides were increased

after 6 and 18 h of MV. RCD accumulation was limited to insoluble proteins with

molecular masses of -200, 120, 80, and 40 kDa. Oxidative stress is therefore evident in

the diaphragm following MV, and is a potential mediator of MV-induced diaphragmatic

dysfunction by way of increased protein degradation.

Skeletal Muscle Unloading and Protein Degradation

In skeletal muscle, the balance between protein synthesis and protein degradation

determines whether muscle growth or atrophy will occur (Baracos et al. 1986a, 1986b).

Protein degradation, specifically myofibrillar protein degradation, may lead to the

dysfunction seen in the diaphragm after MV. Three major pathways exist for general

protein degradation: lysosomal proteases, calcium-activated calpains, and the proteasome

complex. Many studies indicate that the proteasome is responsible for -70-80% of the

increased cellular protein degradation following an oxidative stress (Grune et al.

1995,1996, Grune and Davies 1997). More specifically, it appears the 20S proteasome is

responsible for the degradation of oxidized proteins since the 26S proteasome is

inhibited/inactivated by oxidative stress (Reinheckel et al. 1998). Recognition of exposed

hydrophobic patches is the proposed mechanism by which the proteasome selectively

degrades oxidatively modified proteins (Grune et al. 1997).Oxidative damage to a protein










leads to partial unfolding and exposure of normally shielded internal hydrophobic patches

that are recognized by the proteasome, which catalyzes the degradation of that protein.

Net protein degradation can be estimated from the rate of release of free tyrosine

from tissue proteins (Lowell et al. 1986). Tyrosine is neither synthesized nor degraded in

skeletal muscle, so the net accumulation of this amino acid is directly related to net

degradation of cell protein. Tyrosine and 3-methyl histidine (3-MH) release have been

used to assess total protein degradation and myofibrillar protein degradation,

respectively, under a variety of treatments. For example, Lowell et al. (1986) used both

techniques to identify a differential breakdown of myofibrillar and nonmyofibrillar

proteins during starvation. Likewise, using a model of denervation atrophy, Furuno et al.

(1990) have shown that overall protein breakdown is greater in denervated solei than in

contralateral controls. Treatments that block the lysosomal and Ca2+-dependent

proteolytic pathways did not attenuate protein breakdown, suggesting proteasome-

dependent proteolysis may account for denervation-induced loss of protein. Lastly,

muscle length appears important in determining rates of protein degradation (Baracos et

al. 1986a). Muscles fixed at resting length (Lo) in situ experienced the lowest rate of

protein breakdown compared to unrestrained muscles. The unrestrained solei and EDLs

shortened spontaneously and had 25-45% greater net protein degradation than muscles

fixed at Lo.

Nine days of hindlimb suspension lead to atrophy (-55%), loss of protein (-53%),

and elevated protein breakdown (+66%) in rat soleus muscles compared to controls

(Taillandier et al. 1996). A non-lysosomal, Ca2+-independent proteolytic pathway

accounted for the increased proteolysis and muscle atrophy. This study suggests that










ATP-ubiquitin-dependent proteolysis due to the proteasomal pathway is responsible for

the majority of the increased protein degradation and muscle atrophy in unweighted

hindlimb muscle.

Eighteen h of MV resulted in significant reductions in diaphragmatic protein

content, and significant increases in total in vitro protein degradation, as measured by the

rate of tyrosine release from diaphragm strips (Shanely et al. 2002). The rate of

diaphragmatic protein degradation was increased by 28% after MV compared to controls.

The significant increase in diaphragmatic proteolysis following MV could be reduced

following the addition of either a proteasome inhibitor (lactacystin) or an inhibitor of

both calpain and lysosomal proteases (E64d). These researchers also found that oxidative

stress was elevated in the diaphragm following 18 h of MV. Therefore, MV results in an

increase in oxidative stress (Shanely et al. 2002) that leads to the oxidation of proteins,

making them more susceptible to proteolytic attack and degradation (Grune et al. 1995,

Grune et al. 1996, Nagasawa et al. 1997).

Antioxidant Supplementation and Skeletal Muscle

Muscle cells contain complex defense mechanisms to protect against oxidative

stress (Powers and Hamilton 1999). The two classes of endogenous protective

mechanisms are: 1) enzymatic and 2) nonenzymatic antioxidants. Important enzymatic

antioxidants include superoxide dismutase (SOD), glutathione peroxidase (GPx), and

catalase (CAT). These are responsible for removing superoxide radicals, hydrogen

peroxide or organic hydroperoxides, and hydrogen peroxide, respectively. Important

nonenzymatic antioxidants include vitamins E and C, beta-carotene, glutathione (GSH),

and ubiquinone.












In vitro experiments using excised animal muscle have shown that addition of

antioxidants can delay fatigue and improve muscular performance (Shindoh et al. 1990,

Reid et al. 1992). For example, N-acetylcysteine (NAC) has been shown to protect an in

situ rabbit diaphragm strip preparation from oxidative injury during periods of rhythmic

repetitive isometric contraction (Shindoh et al. 1990). The researchers postulate that

NAC, a potent radical scavenger, may have affected fatigue by preventing free radical-

mediated damage in the exercising diaphragm muscle. Similarly, Reid et al. (1992) found

improved muscular performance in rat diaphragm fiber bundles with both SOD and CAT

supplementation. These antioxidants inhibited low-frequency fatigue, but did not alter

high-frequency fatigue.

The effects of antioxidant supplementation on human performance are less

definitive. Many studies using human subjects have experimental design weaknesses, and

most have only investigated the effects of a single antioxidant rather that combining both

lipid-soluble and water-soluble antioxidants (Powers and Hamilton 1999). Few studies

show improved human exercise performance with antioxidant supplementation.

However, the laboratory of Reid and colleagues (1994) has shown that NAC

administration in human subjects improves muscular endurance during low-frequency

electric stimulation. During fatiguing contractions at 10 Hz, NAC increased force

production by -15%. However, NAC had no effect on fatigue induced by 40 Hz

stimulation, or on recovery from fatigue. Additional research is required to determine the

specific effects of supplemental antioxidants on humans. Careful research design and

understanding of bioavailability are essential to draw conclusions.










Vitamin E

The most widely studied antioxidant is vitamin E, or alpha-tocopherol (Powers and

Hamilton 1999). Kondo et al. (1993a) injected either vitamin E or placebo one time daily

into Wistar rats with one hindlimb immobilized for 7 days with the soleus muscle in a

shortened position. The TBARS level of atrophic muscle in the vitamin E group was

significantly less than in the placebo group. The muscle weight was significantly greater,

and the degree of atrophy was significantly reduced by -20% in the vitamin E group

compared to the placebo group. Intraperitoneal injections of vitamin E during periods of

muscle atrophy effectively served as an antioxidant to reduce oxidative stress and prevent

muscle atrophy.

Eight days of immobilization led to a 35% atrophy in the hindlimb of rats (Appell

et al. 1997). However, when vitamin E was supplemented, the muscles atrophied by only

12%. Control muscles of those animals supplemented with vitamin E contained even less

of the oxidized form of glutathione (GSSG) than baseline oxidative stress. These results

indicate that the soleus muscle atrophies to a lesser extent when supplemental vitamin E

is given during a period of disuse.

Trolox

Vitamin E is a natural antioxidant, but is extremely lipophilic and is taken up

slowly by cells (Zeng et al. 1991). It is therefore not an adequate therapeutic antioxidant.

Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), is a hydrophilic

vitamin E analog synthesized in 1974 by Scott and colleagues (1974). Trolox differs from

vitamin E by the absence of the phytyl side chain, which makes Trolox water-soluble

(Klein et al. 1991).













Trolox is an effective scavenger of radicals (Walker et al. 1998). This antioxidant

has been shown to prolong the survival of cells exposed to oxyradicals (Wu et al. 1990,

Wu et al. 1991). Specifically, it was found that Trolox protected human ventricular

myocytes and hepatocytes against oxyradicals generated by xanthine oxidase-

hypoxanthine and prevented lysis of erythrocytes exposed to an azo-inhibitor (Wu et al.

1990). The protection by Trolox was dose dependent in all cell types, and surpassed the

antioxidant capabilities of ascorbic acid, SOD, and CAT. Using hepatocytes, the

researchers determined that Trolox behaved mechanistically as an antioxidant in cells.

In cultured rat hepatocytes, 0.5-16 mmol/L Trolox prolonged the survival of cells

exposed to xanthine oxidase-hypoxanthine oxyradicals (Wu et al. 1991). Optimum levels

of Trolox were between 1 and 2 mmol/L. Protection by Trolox surpassed that provided

by ascorbate, mannitol, SOD, and CAT. This laboratory also studied a global and partial

model of hepatic ischemia-reperfusion in rats (Wu et al. 1991). Infusion of Trolox (7.5-10

[tmol/kg body weight) prior to reflow reduced liver necrosis by more than 80% compared

to control, untreated animals. These data indicate a strong and rapid antioxidant-like

action by Trolox on rat hepatocytes and postischemic-reperfused rat liver.

Trolox also protected regionally ischemic, reperfused porcine hearts against free

radical generation (Klein et al. 1991). Specifically, Trolox reduced free radical generation

from stimulated neutrophils by 30% in the treatment group before ischemia and

immediately before reperfusion. After 3 days of reperfusion, recovery of regional

function had improved to a significantly greater extent in the treated group than in control

hearts. Mean recovery of systolic shortening amounted to 10% of the baseline value in









the control animals, and to 28% in the Trolox group. Trolox did not reduce infarct size,

but did accelerate functional recovery in ischemic, reperfused porcine hearts.

Trolox has also been shown to improve the long-term storage of isolated skeletal

muscle (van der Heij den et al. 2000). Significant protection of contractile function

occurred with addition of ImM Trolox in the bathing solution of soleus and cutaneus

trunci muscles from the rat. These muscles were stored for 16 h at 40C. Trolox effectively

reduced the overproduction of oxyradicals.

Trolox treatment in vivo protected methylmercury (MeHg)-treated rat skeletal

muscle from many of the clinical manifestations of MeHg-intoxication (Usuki et al.

2001). Trolox prevented decreases in mitochondrial enzyme activities in soleus muscle,

repressed apoptosis in cerebellum, and protected against the decrease in glutathione

peroxidase activity of the soleus following MeHg-intoxication.

Summary

The removal of weight bearing from skeletal muscle leads to rapid and significant

atrophy. The rat model of mechanical ventilation effectively unloads the diaphragm

muscle, thereby causing atrophy. Oxidative stress is evident in unloaded muscle,

including diaphragm muscle from mechanically ventilated animals, and may increase

rates of protein degradation. Interventions with antioxidants have shown that muscle

atrophy and dysfunction can be attenuated during unloading. The antioxidant properties

of Trolox make it an appealing subject for research. No studies have given Trolox to

animals to prevent muscle dysfunction during unloading. This project will determine

whether mechanical ventilation-induced diaphragmatic dysfunction, oxidative stress, and

proteolysis can be attenuated with supplementation of the antioxidant-like compound,

Trolox.














CHAPTER 3
METHODS

Experimental Design

The following groups were formed to complete these experiments:

Pure control group CON n=8
Spontaneous breathers SBS n=8
Mechanical ventilation MVS n=8
Spontaneous breathers SBT n=3
receiving Trolox
Mechanical ventilation MVT n=8
receiving Trolox

Animals

The subjects were adult (-4 month-old) female Sprague-Dawley rats (-250 g). All

were housed in the J. Hillis Miller Animal Science Center and fed the same diet (rat chow

and water ad libitum) for one week prior to the experiment. Animals were maintained on

a 12 h light:dark photoperiod. All procedures followed NIH guidelines and were

approved by the University of Florida's Animal Care and Use Committee.

General Procedures

After a period of acclimation (1 week), rats were randomly assigned to one of the 5

groups listed above. The control group (CON) animals were free of intervention before

removal of the diaphragm for measurements. These animals received an acute

intraperitoneal injection of sodium pentobarbital (65 mg/kg body weight). When a

surgical plane of anesthesia was reached, the diaphragms were removed for measurement

of in vitro contractile properties and tyrosine release, and remaining muscle was frozen

for biochemical assays.









Animals in the 2 mechanically ventilated groups (MVS and MVT) were given an

intramuscular injection of glycopyrollate (0.04 mg/kg) to reduce respiratory secretions

during the protocol. Thirty min later, animals were anesthetized with an intraperitoneal

injection of sodium pentobarbital (65 mg/kg). Upon reaching a surgical plane of

anesthesia (no ocular response, no hindlimb withdrawal response), they were

tracheostomized by an experienced lab technician and mechanically ventilated with a

volume-cycled ventilator (Harvard Apparatus). The tidal volume was established at -1

ml/100 g body weight with a respiratory rate of 80 breaths/min. Positive end-expiratory

pressure of 1 cm H20 was used for all MV animals. Throughout the MV period, heart

activity, blood pressure, and core temperature were monitored. A lead II ECG displayed

electrical activity of the heart. A catheter placed in the carotid artery gave constant blood

pressure readings. Core temperature was monitored with a rectal thermometer and

adjustments were made to help animals maintain body temperature at 37 + 1C with a re-

circulating heating blanket.

A catheter was placed in the jugular vein for the infusion of sodium pentobarbital

(-10 mg/kg/h) and Trolox. In the MVT group, the jugular vein was cannulated before the

carotid artery and before the tracheotomy. A priming dose of Trolox (20 mg/kg) was

infused over a 5 min period. Twenty min later, MV was started, along with the constant

infusion of Trolox at a rate of 5 mg/kg/h. Constant supervision was provided for the rats

throughout the MV period. This included expressing the bladder, removing airway

mucus, monitoring anesthesia rate, rotating the animals, and infusing saline to maintain

hydration status. To reduce airway secretions, glycopyrollate (0.04 mg/kg) was injected

intramuscularly every 2 h.









Spontaneously breathing (SBS and SBT) animals were anesthetized in the same

manner and received sham surgeries. These animals were included in the study to

determine whether long-term anesthesia (sodium pentobarbital) impairs diaphragmatic

contractile function. A tube was inserted into the trachea, and these animals were

maintained on a surgical plane of anesthesia for the 12 h period while continuing to

breathe on their own. The carotid artery and jugular vein were cannulated, and sodium

pentobarbital was infused for a 12 h period. However, these animals were not

mechanically ventilated. The SBT group received the same dose of Trolox as the MVT

group (20 mg/kg priming dose, 5 mg/kg/h constant infusion).

At the end of the 12 h experimental period, all rats were killed by injection of

sodium pentobarbital (50 mg/kg) and the diaphragm was removed for immediate analyses

of contractile function and protein degradation as described below. After obtaining

muscle strips for contractile and protein degradation measurements, the remaining costal

diaphragm tissue was dissected, weighed, frozen in liquid nitrogen, and stored at -80C

until needed.

Diaphragm Contractile Function

The entire diaphragm with the supporting ribs and central tendon was removed and

placed in a dissecting chamber containing a Krebs-Hensleit solution aerated with 95%

02-5% CO2 gas. The entire crural diaphragm was removed and discarded. A strip was cut

from the midcostal region including the central tendon and the rib. This was secured

vertically in an organ bath maintained at 24C between two Plexiglass clamps. The

muscle strip was placed between two platinum field electrodes connected to an isometric

force transducer (model FT-03, Grass Instruments, Quincy, MA). This strip was mounted

on a micrometer to allow for muscle length adjustment. A 15 min equilibration period in










the bath preceded all data collection. During this time, the remaining diaphragm muscle

was dissected and sectioned into 9 pieces: 2 dorsal, 6 midcostal, and 1 ventral section. All

sections were blotted, weighed, frozen in liquid nitrogen, and stored at -800C. All further

analyses were conducted on midcostal diaphragm sections.

We determined optimal muscle length (Lo), the length that generates maximal

twitch force, and used this length throughout the protocol. Lo was found by

systematically adjusting the length of the muscle while stimulating it with single

supramaximal (-150%) twitches and recording the force generated. Lo was measured (in

cm) using calipers.

Peak isometric tetanic tension was measured from a series of three contractions

with 2 min of recovery between contractions. The force-frequency relationship was

studied by stimulating the muscle strips at 15, 30, 60, 100, 160, and 200 Hz (120 V).

Each stimulus was applied for 500 ms, and adjacent stimulus trains were separated by 2

min of rest.

Diaphragmatic fatigability was assessed by monitoring the decrease in force

development over time. Each muscle strip was stimulated by unfused tetanic contractions

(30 Hz, 250 ms) for 30 min. The duty cycle, or time of muscle contraction compared to

muscle rest, was 12.5%. Tension was measured at 0, 1, 2, 5, 10, 15, 20, 25, and 30 min.

Fatigue resistance was assessed by the percentage of initial force maintained at the end of

the 30 min protocol. After all contractile measurements were made, the diaphragm strip

was removed from the organ bath. The rib, central tendon, and excess fat and connective

tissue were removed from the strip, which was then blotted and weighed. Forces

generated were normalized to muscle strip cross-sectional area (CSA).









Protein Degradation

To measure total protein degradation, the release of tyrosine into the incubation

medium was measured. Two strips were cut from the midcostal diaphragm (- 40 mg

each). These strips were secured at resting length in separate baths containing Krebs-

Ringer bicarbonate solution, which was supplemented with 5 mM glucose, insulin (1

unit/ml), 0.17 mM leucine, 0.10 mM isoleucine, and 0.20 mM valine to improve protein

balance, and 5 mM cycloheximide to inhibit protein synthesis. Diaphragm strips were

maintained at resting lengths by securing both ends to a solid plexiglass rod. The medium

was continuously gassed with 95% 02 5% CO2. Temperature was maintained at 370C.

Muscle strips were preincubated for 30 min, and then fresh medium was added for a 2 h

incubation. After this incubation, the strips were removed, blotted, and weighed. For

measurement of tyrosine release, the medium from each bath was aliquoted into

microcentrifuge tubes. The aliquots were stored at -200C until analysis of tyrosine

concentration.

Tyrosine in the medium was assayed spectrofluorometrically by the method of

Waalkes and Udenfriend (1957) with some modification. Two hundred [l of incubation

medium was diluted with 800 al dH20 in a glass tube. To this, 0.5 ml of 1-nitroso-2-

naphthol reagent (0.1 g 1-nitroso-2-naphthol in 100 ml of 95% methanol) and 0.5 ml of

nitric acid reagent (24.5 ml of 20% nitric acid and 0.5 ml of 2.5% NaNO2) were added.

The tubes were shaken to mix, and incubated in a water bath at 55C for 30 min. After

cooling for 15 min, 5.0 ml of ethylene dichloride was added to extract the unchanged

nitrosonaphthol reagent. The tubes were centrifuged for 15 min at 2500 x g. One ml of

the supernatant was transferred to a quartz cuvette and read in a spectrophotofluorometer.









The tyrosine derivative was excited at 460 uM and measured at 570 uM. Standards were

prepared using L-tyrosine (Sigma).

20S Proteasome Activity

The chymotrypsin-like activity of the 20S proteasome was measured

fluorometrically as the release of AMC from the synthetic substrate Suc-LLVY-AMC.

Approximately 50 mg of midcostal diaphragm tissue was homogenized (glass-on-glass)

in a homogenizing buffer containing 50 mM Tris base, 1 mM EDTA, 1 mM EGTA, 1

tM Pepstatin-A, 50 tM E-64, and 10% glycerol. This homogenate was centrifuged at

1500 x g for 10 min at 40C, and the supernatant was then centrifuged at 10,000 x g for 10

min at 40C. The remaining supernatant was centrifuged at 100,000 x g in an

ultracentrifuge for 1 h at 40C to separate the proteasomal fraction. The resulting

supernatant fraction was used to measure protein content using the Bradford method, and

to measure proteasome activity. Ten tg of protein was reacted with the synthetic peptide

substrate for chymotrypsin-like activity in a reaction mixture containing 50mM Tris-HC1,

1 mM DTT, and 5 mM MgCl2. One aliquot from each sample was incubated with an

inhibitor of the chymotrypsin-like proteasomal activity, lactacystin, while the other was

not. Samples were incubated for 30 min at 370C before the addition of substrate. The

change in fluorescence was measured at an excitation wavelength of 380 nM and

emission of 460 nM. The difference between the activities of the proteasome with and

without inhibitor was used as the proteasome activity.

Total and Non-protein Thiols

As an indicator of oxidative stress, we measured total thiol and non-protein thiol

groups in diaphragm homogenate. Diaphragm tissue was homogenized in 0.02 M EDTA

on ice and centrifuged at 1500 rpm for 10 min at 5 C. Twenty-five pl of homogenate was










incubated with 75 [l 0.2 M Tris buffer (pH 8.2), 395 [l methanol, and 5 [l 0.01 M DTNB

for 35 min on a bench-top rotator. Samples were centrifuged at room temperature for 15

min at 3,000 x g. Two hundred [l of supernatant was loaded per microplate well (3 wells

per sample) and read at a wavelength of 414 nm. A standard curve was generated using

glutathione (GSH; Sigma) in 0.02 M EDTA.

Non-protein thiols were measured by incubating 350 ul homogenate with 350 ul

1% metaphosphoric acid for 15 min on a bench-top rotator to precipitate proteins.

Samples were centrifuged at room temperature for 15 min at 3,000 x g. Three hundred ul

supernatant was then incubated with 200 ul 0.4 M Tris buffer (pH 8.9) and 25 ul 0.01 M

DTNB. Tubes were mixed for 10 min and read against GSH standards in a microplate

reader at 414 nm.

Limitations

We did not record electromyographic (EMG) activity of the mechanically

ventilated diaphragms to ensure that muscle activation was completely suppressed.

Powers et al. (2002) performed preliminary experiments where wire electrodes that

measure EMG activity were placed in the costal diaphragm of 4 animals during 24 h of

controlled MV. No electrical activity was measured in any of these animals during the 24

h procedure. Le Bourdelles and colleagues (1994) did not find EMG activity in the rat

diaphragm ventilated for 2 days. Therefore, we assumed that diaphragmatic contractions

did not occur during 12 h of controlled MV.

The mortality rate of the SBT group was higher than other groups (-70%). As a

result, we were only able to include 3 animals that survived the 12-h spontaneous

breathing protocol while receiving Trolox. This made statistical analyses difficult, and

conclusions yet uncertain with this group.









Vertebrate Animals

Female Sprague-Dawley rats were used in this research. This study required

removal of the diaphragm muscle for analysis, and therefore prevented the use of human

subjects. Sprague-Dawley rats were selected since our lab, as well as previous

researchers, has successfully used them as subjects in MV studies. The MV experiments

were supervised by research assistants who have experience with short-term MV in rats.

Statistical Analysis

This experiment was designed to test the hypotheses that Trolox supplementation

during mechanical ventilation would alter diaphragm contractile dysfunction, protein

degradation, and oxidative stress. A 4 x 6 (group x stimulation frequency) ANOVA with

repeated measures on stimulation frequency was used to analyze the force-frequency

data. Likewise, a 4 x 9 (group x time) ANOVA with repeated measures on the time factor

was used to analyze data from the fatigue protocol. Where significant differences were

found, Tukey's HSD test was implemented post hoc. ANOVAs were used to examine

differences between groups for the remaining dependent variables. Independently, the

effects of anesthesia were compared using a Student's t-test on SBS and CON group data.

Significance was established at p<0.05.














CHAPTER 4
RESULTS

Systemic and Biologic Responses to Treatment

The MV protocol did not significantly change body mass for any of the groups

(Table 1), indicating that our schedule of nutrition and rehydration was adequate. The

ratio of total costal diaphragm mass to final body mass was not significantly different

between the 5 groups (p=0.501).

There were no signs of infection in any animals, and only 1 MVS animal was

eliminated from the study due to evidence of barotrauma to the lungs on post-mortem

examination. Systolic blood pressure was maintained at 70-110 mmHg in all groups, and

arterial pH, PO2, and PCO2 were maintained within physiological ranges for both MV

groups. The SB animals were mildly hypoxic, hypercapnic, and acidotic, as expected due

to the anesthesia. Body temperature was kept at 37 + 1C during the 12-hour protocol.

Effects of Anesthesia on Diaphragm Contractile Properties

The maximal tetanic force was not different between the SBS and CON groups

(25.09 + 0.41 N/cm2 vs. 25.33 + 0.50 N/cm2, respectively). Likewise, the force-frequency

curves and fatigue data were similar between these two groups (Figures 1, 2, and 3), and

contractile parameters did not differ (Table 3). Thus, 12 hours of sodium pentobarbital

anesthesia did not affect in vitro contractile properties of the diaphragm.

Effects of Mechanical Ventilation on Contractile Properties

Twelve hours of controlled MV reduced maximal tetanic force production by -17%

(21.00 + 0.71 N/cm2 vs. 25.43 + 0.50 N/cm2 in CON animals). The force-frequency curve









of the MVS group was shifted downward and to the right of the CON group (Figure 1).

This indicates a reduction in force generation at all stimulation frequencies tested. The

fatigue protocol produced curves of similar shape for all groups (Figure 2), but the MVS

group generated a significantly lower amount of force compared to CON, SBS, and MVT

groups where indicated. When the fatigue data are expressed as percent of initial force

(Figure 3), there are no significant differences between the 4 groups at any time point (p=

0.230).

One-half relaxation time (/2 RT) of maximal twitch was significantly shorter in the

MVS group compared to CON, while /2 time to peak tension (/2 TPT), rate of force

development, and rate of relaxation were not different for either maximal twitch or

maximal tetanic forces (Table 3).

Effects of Trolox on Contractile Properties

Trolox supplementation during 12 hours of MV completely attenuated the loss of

maximal force generation. The MVT group was not significantly different from CON at

any stimulation frequency tested (Figure 1). Animals receiving Trolox during MV

maintained a greater force generating ability following the fatigue protocol compared to

the unsupplemented MVS group (Figure 2). Trolox during MV significantly prolonged

the rate of relaxation of maximal twitch compared to CON, but did not affect other

contractile parameters (Table 3).

Twelve hours of Trolox infusion without MV (i.e. the SBT group) greatly increased

subject mortality rate. Only three SBT animals survived the treatment. Further,

diaphragms from these animals showed impaired force production compared to CON.

The force-frequency curve of the SBT group was shifted downward and to the right of

the CON group and closely resembles the MVS group (Figure 1). Trolox










supplementation during spontaneous breathing also significantly reduced /2 TPT of

maximal tetanic force compared to CON animals.

Protein Degradation

Twelve hours of controlled MV significantly elevated total in vitro protein

degradation (+105%), as measured by the release of free tyrosine, compared to CON

(Figure 4). However, protein degradation of the MVT group (16% increase compared to

CON) was not significantly different from CON (p=0.797). There were no significant

differences in protein degradation between CON and SBS groups (p=0.351).

20S Proteasome Activity

The chymotrypsin-like activity of the 20S proteasome was significantly increased

in the MVS group compared to the CON group (+76%) (Figure 5). Trolox attenuated the

MV-induced increase in proteasome activity (+26% compared to CON, p=0.647).

Oxidative Stress

Protein carbonyls and lipid hydroperoxides, two indicators of oxidative stress, were

not different between CON, MVS, and MVT groups. However, total thiols were

significantly lower in the MVS group as compared to the CON group (134.83 + 6.90

nmol/mg protein vs. 157.34 + 6.45 nmol/mg,respectively). Similarly, non-protein thiols

were significantly lower in the MVS group compared to the CON group (28.02 + 1.62

nmol/mg vs. 42.14 + 1.51 nmol/mg, respectively). But, Trolox supplementation during

MV failed to prevent the loss of total and non-protein thiol groups.










Table 4-1. Body and Diaphragm Weights of Control, Spontaneously Breathing, and
Mechanically Ventilated Animals

CON SBS MVS SBT MVT
Initial body mass (g) 264.38 + 5.16 276.88 + 5.29 282.25 + 3.33 285.67 + 2.33 300.13 + 6.37*
Final body mass (g) 264.38 + 5.16 280.56 + 5.52 286.38 + 3.18* 292.00 + 2.00 306.75 + 6.42*
Total costal diaphragm 532.57 + 15.64 567.69 + 12.86 601.86 + 14.80 610.63 + 27.98 632.98 + 10.74
mass (mg)
Total costal diaphragm 2.014 + 0.04 2.025 + 0.04 2.100 + 0.04 2.091 + 0.09 2.067 + 0.04
mass/body massT
(mg/g)

Definition of abbreviations: CON = control animals; SBS = spontaneously breathing animals receiving
saline; MVS = mechanically ventilated animals receiving saline; SBT = spontaneously breathing animals
receiving Trolox; MVT = mechanically ventilated animals receiving Trolox.
Values represent means + SEM
Significantly different from CON group, p<0.05.
t Significantly different from SBS group, p<0.05.
t Mass values expressed as milligrams per gram of body mass were normalized to
postexperiment body mass values.


Table 4-2. Maximal Isometric Twitch and Tetanic Force of Control, Spontaneously
Breathing, and Mechanically Ventilated Animals

CON SBS MVS SBT MVT
Maximal
isometric twitch 7.24 + 0.14 6.79 + 0.30 5.65 + 0.28*t 5.94 + 0.55 7.04 + 0.25
force (N c:m )
Maximal
isometric tetanic 25.43 + 0.50 25.09 + 0.41 21.01 + 0.71*t 21.54 + 1.40*t 25.49 + 0.50
force (N m )____________________

Definition of abbreviations: CON = control animals; SBS = spontaneously breathing animals receiving
saline; MVS = mechanically ventilated animals receiving saline; SBT = spontaneously breathing animals
receiving Trolox; MVT = mechanically ventilated animals receiving Trolox.
Values represent means + SEM
Significantly different from CON group, p<0.05.
t Significantly different from SBS group, p<0.05.
T Significantly different from MVT group, p<0.05.










Table 4-3. Contractile Parameters of Maximal Isometric Twitch and Tetanic Forces of
Control, Spontaneously Breathing, and Mechanically Ventilated Animals

CON SBS MVS SBT MVT


TWITCH
/2 TPT 0.018 + 0.000 0.018 + 0.001 0.017 + 0.000 0.017 + 0.000 0.018 + 0.000
12 RT 0.044 + 0.001 0.040 + 0.001 0.034 + 0.003* 0.037 + 0.002 0.04 + 0.001
+ dp/dt 332.29 + 27.368 420.97 + 31.176 415.85 + 57.784 418.82 + 94.619 459.52 + 18.567
dp/dt -149.26 + 9.247 -177.84 + 9.696 -207.93 + 28.890 -179.61+ 34.138 -204.21 + 7.845*

TETANIC
S/2TPT 0.064 + 0.002 0.062 + 0.001 0.056 + 0.003 0.053 + 0.003* 0.059 + 0.002
/2 RT 0.064 + 0.002 0.062 + 0.003 0.062 + 0.003 0.061 + 0.001 0.065 + 0.002
+ dp/dt 423.71 + 34.738 527.67 + 41.175 515.38 + 95.654 518.80 + 93.776 570.18 + 20.668
dp/dt -544.55 + 50.860 -723.04 + 36.225 -703.53 + 157.300 -658.41 + 94.133 -740.20 + 12.091

Definition of abbreviations: CON = control animals; SBS = spontaneously breathing animals receiving
saline; MVS = mechanically ventilated animals receiving saline; SBT = spontaneously breathing animals
receiving Trolox; MVT = mechanically ventilated animals receiving Trolox; TPT = time to peak tension;
RT = relaxation time; +dp/dt = rate of force development; -dp/dt= rate of force relaxation.
Values represent means + SEM
* Significantly different from CON group, p<0.05.
t Significantly different from SBS group, p<0.05.
T Significantly different from MVT group, p<0.05.



























26-

24

22 + #

20 -

18-
"/ -*- CON
0 -0- SBS
u.. 16 MVS
MVT
14-

12

10-
25 50 75 100 125 150 175 200

Stimulation Frequency (Hz)


Figure 4-1. Force-frequency responses of control (CON), spontaneously breathing (SBS),
mechanical ventilation (MVS), and mechanical ventilation animals receiving
Trolox (MVT).

Values represent means + SEM.
* Significantly different from CON group, p<0.05.
+ Significantly different from SBS group, p<0.05.
# Significantly different from MVT group, p<0.05.


om SBS group, p<0.05.
# Significantly different from MVT group, p<0.05.













-*- CON
-0- SBS
- MVS
-V- MVT


0 5 10 15 20 25 30
0 5 10 15 20 25 30


Time (min)





Figure 4-2. Responses of in vitro diaphragm strips from control (CON), spontaneously
breathing (SBS), mechanical ventilation (MVS), and mechanical ventilation
animals receiving Trolox (MVT) to a 30-min fatigue protocol.

Values represent means + SEM.
* Significantly different from CON group, p<0.05.
+ Significantly different from SBS group, p<0.05.
# Significantly different from MVT group, p<0.05.















100


-*- CON
-0- SBS
-7- MVS
-v- MVT


40




20 -
0


10 15 20 25 30 35


Time (min)





Figure 4-3. Percent of initial force maintained by in vitro diaphragm strips from control
(CON), spontaneously breathing (SBS), mechanical ventilation (MVS), and
mechanical ventilation animals receiving Trolox (MVT) after a 30-min fatigue
protocol.

Values represent means + SEM.
* Significantly different from CON group, p<0.05.
+ Significantly different from SBS group, p<0.05.
# Significantly different from MVT group, p<0.05.







44












0.6
*,+,#

0.5


(N 0.4

0
E
0.3


S0.2 -
I--

0.1


0.0
CON SBS MVS MVT


Group



Figure 4-4. Total in vitro diaphragmatic protein degradation as measured by the rate of
tyrosine release from control (CON), spontaneously brearhing (SBS),
mechanical ventilation (MVS), and mechanical ventilation animals receiving
Trolox (MVT) during a 2-hour incubation.

Values represent means + SEM.
* Significantly different from CON group, p<0.05.
+ Significantly different from SBS group, p<0.05.
# Significantly different from MVT group, p<0.05.











0.8



>, 0.6-

< 0
() 0-

0--
E
0-
04 0.2



0.0
CON SBS MVS MVT
Group

Figure 4-5. Chymotrypsin-like activity of the 20 S proteasome in diaphragm tissue from
control (CON), spontaneously breathing (SBS), mechanical ventilation
(MVS), and mechanical ventilation animals receiving Trolox (MVT).

Values represent means + SEM.
* Significantly different from CON group, p<0.05.







46




200-



c 180-









1-
o)
E 160-







F- 120



100
CON SBS M3S WF

Q-ap


Figure 4-6. Total thiol concentration in diaphragm tissue from control (CON),
spontaneously breathing (SBS), mechanical ventilation (MVS), and
mechanical ventilation animals receiving Trolox (MVT).

Values represent means + SEM.
* Significantly different from CON group, p<0.05.







47





60 -



S50 -
00







30
0







20
3


10
CON SBS MVS MVT
Group






Figure 4-7. Non-protein thiol concentration in diaphragm tissue from control (CON),
spontaneously breathing (SBS), mechanical ventilation (MVS), and
mechanical ventilation animals receiving Trolox (MVT).

Values represent means + SEM.
* Significantly diffrom t from CON group, p<0.05.
+ Significantly different from SBS group, p<0.05.
# Significantly different from MVS group, p<0.05.














CHAPTER 5
DISCUSSION

Trolox Attenuates Mechanical Ventilation-induced Contractile Dysfunction and
Proteolysis in the Rat Diaphragm: Introduction

Weaning patients from a mechanical ventilator is a serious clinical issue.

Mechanical ventilation (MV) is characteristically used in the clinical setting to maintain

alveolar ventilation in patients who are incapable of ventilation on their own. As such,

MV is an important life-preserving measure, but removing patients from the ventilator

can be difficult in many cases. As many as 20% of patients experience difficulty in

weaning from the ventilator (Lemaire 1993). Weaning procedures account for more than

40% of total MV time in patients who have difficulty weaning (Esteban 1994),

suggesting that this is a serious clinical issue.

Our laboratory has reported that MV-induced diaphragmatic dysfunction is intrinsic

to the muscle and increases in magnitude with increasing time on the ventilator (Powers

et al. 2002). However, the mechanisms) behind MV-induced diaphragmatic atrophy and

weakness remain unclear. Since oxidative stress has been linked to reduced-use atrophy

(Kondo et al. 1993), and protease-mediated protein degradation in unloaded locomotor

muscle (Taillandier et al. 1996), we examined markers of oxidative stress during MV.

Eighteen hours of controlled MV elevated markers of oxidative stress such as protein

carbonyls and 8-isoprostanes (Shanely et al. 2002). This is significant because oxidized

proteins are more prone to proteolytic attack and degradation (Dean 1997, Nagasawa et

al. 1997). Indeed, 18 hours of MV is associated with an increase in protein degradation









through increases in total calpain-like activity and 20S proteasome activity (Shanely et al.

2002). Therefore, it seems logical that diaphragmatic weakening during MV-induced

unloading may be caused by oxidative damage leading to heightened proteolytic

degradation.

The purpose of this study was to determine whether supplementation with Trolox

(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) during 12 hours of controlled

MV would attenuate diaphragmatic contractile dysfunction, reduce oxidative stress, and

attenuate protein degradation. Trolox is a water-soluble vitamin E analog with

antioxidant properties (Wu et al. 1990, Zeng et al. 1991, Walker et al. 1998). We

hypothesized that Trolox would maintain redox balance within the muscle by functioning

as an antioxidant, and thereby prevent oxidative stress, and subsequent proteolysis and

contractile dysfunction.

Materials and Methods

Animals and Experimental Design

Female Sprague-Dawley rats (-250 g) were obtained from Harlan (Indianapolis,

IN). They were maintained on a 12-hour light:dark photoperiod and fed rat chow and

water ad libitum prior to initiation of experiments. Animals were randomly assigned to

one of five groups: (1) Controls receiving acute anesthesia and no further intervention

(CON, n=8) (2) 12 hours of mechanical ventilation (MVS, n=8), (3) 12 hours of MV and

Trolox infusion (MVT, n=8), (4) 12 hours of anesthesia and spontaneous breathing (SBS,

n=8), and (5) 12 hours of anesthesia and spontaneous breathing with Trolox infusion

(SBT, n=3). All procedures were approved by the University of Florida Animal Care and

Use Committee.









Control Animal Protocol

The control animals (CON) were free of intervention before removal of the

diaphragm for measurements. These animals received an acute intraperitoneal injection

of sodium pentobarbital (65 mg/kg body weight). When a surgical plane of anesthesia

was reached, the diaphragms were removed for measurement of in vitro contractile

properties and tyrosine release, and remaining muscle was weighed and frozen for

biochemical assays.

Mechanical Ventilation Protocol

Animals in the mechanically ventilated groups (MVS and MVT) were given an

intramuscular injection of glycopyrollate (0.04 mg/kg) to reduce respiratory secretions.

Thirty minutes later, subjects were anesthetized with an intraperitoneal injection of

sodium pentobarbital (65 mg/kg). Upon reaching a surgical plane of anesthesia (no ocular

response, no hindlimb withdrawal response), they were tracheostomized by an

experienced lab technician and mechanically ventilated with a volume-cycled ventilator

(Harvard Apparatus). The tidal volume was established at ~1 ml/100 g body weight with

a respiratory rate of 80 breaths/min. Positive end-expiratory pressure of 1 cm H20 was

used for all ventilated animals. Throughout the MV period, heart activity, blood pressure,

and core temperature were monitored. A lead II ECG displayed electrical activity of the

heart. A catheter was placed in the carotid artery for constant blood pressure readings and

arterial blood sampling. Core temperature was monitored with a rectal thermometer and

adjustments were made to maintain body temperature at 37 1C with a re-circulating

heating blanket.

A catheter was placed in the jugular vein for the infusion of sodium pentobarbital

(-10 mg/kg/h) and Trolox (Fluka). Constant supervision was provided for the rats









throughout the MV period. This included expressing the bladder, removing airway

mucus, providing enteral nutrition, monitoring anesthesia rate, rotating the animals,

lubricating the eyes, and infusing saline to maintain hydration status. To reduce airway

secretions, glycopyrollate (0.04 mg/kg) was injected intramuscularly every 2 hours.

In the MVT group, the jugular vein was cannulated first. A priming dose of Trolox

(20 mg/kg) was infused over a 5 min period. Twenty min later, MV was started, along

with the constant infusion of Trolox at a rate of 5 mg/kg/h.

At the end of the 12-hour experimental period, all rats were killed by injection of

sodium pentobarbital (50 mg/kg) and the diaphragm was removed for immediate analyses

of contractile function and protein degradation as described below. Remaining costal

diaphragm tissue was dissected, weighed, frozen, and stored for biochemical analyses.

Spontaneous Breathing Protocol

Spontaneously breathing (SB) animals were anesthetized in the same manner as

MV animals and received sham surgeries. These animals were included in the study to

determine whether long-term anesthesia (sodium pentobarbital) impairs diaphragmatic

contractile function. A tube was inserted into the trachea, and these animals were

maintained on a surgical plane of anesthesia for the 12-hour period while continuing to

breathe on their own. The carotid artery and jugular vein were cannulated, and sodium

pentobarbital was infused for the 12 hours. However, these animals were not

mechanically ventilated.

The SBT group received the same dose of Trolox as the MVT group (20 mg/kg

priming dose, 5 mg/kg/h constant infusion) for the 12-hour experimental period. The

same general care was provided for these animals as for MV animals.









Contractile Measurements

The entire diaphragm with the supporting ribs and central tendon was removed and

placed in a dissecting chamber containing a Krebs-Hensleit solution aerated with 95%

02-5% CO2 gas. The entire crural diaphragm was removed and discarded. A strip was cut

from the midcostal region including the central tendon and the rib. This was secured

vertically in an organ bath maintained at 240C between two Plexiglass clamps. The

muscle strip was placed between two platinum field electrodes connected to an isometric

force transducer (model FT-03, Grass Instruments, Quincy, MA). This strip was mounted

on a micrometer to allow for muscle length adjustment. A 15 min equilibration period in

the bath preceded all data collection. During this time, the remaining diaphragm muscle

was dissected and sectioned. All sections were weighed, frozen in liquid nitrogen, and

stored at -800C for further analyses.

We determined optimal muscle length (Lo), the length that generates maximal

twitch force, and used this length throughout the protocol. Lo was found by systematically

adjusting the length of the muscle while stimulating it with single supramaximal (-150%)

twitches and recording the force generated. Lo was measured using calipers.

The force-frequency relationship was studied by stimulating the muscle strips at 15,

30, 60, 100, 160, and 200 Hz (120 V). Each stimulus was applied for 500 ms, and

adjacent stimulus trains were separated by 2 min of rest. Peak isometric tetanic tension

was determined from these measurements.

Diaphragmatic fatigability was assessed by monitoring the decrease in force

development over time. Each muscle strip was stimulated with unfused tetanic

contractions (30 Hz, 250 ms) for 30 min. The duty cycle, or time of muscle contraction

compared to muscle rest, was 12.5%. Tension was measured at 0, 1, 2, 5, 10, 15, 20, 25,









and 30 min. Fatigue resistance was assessed by the percentage of initial force maintained

at the end of the 30 min protocol. After all contractile measurements were taken, the

diaphragm strip was removed from the organ bath. The rib, central tendon, and excess fat

and connective tissue were removed from the strip, which was then weighed. Forces

generated were normalized to muscle strip cross-sectional area (CSA), calculated from

strip weight and length at Lo.

Protein Degradation

To measure total in vitro protein degradation, the release of free tyrosine into the

incubation media was assayed. The rationale for this technique is that tyrosine is neither

synthesized nor degraded by skeletal muscle, making it an ideal marker of total muscle

protein breakdown (Tischler et al. 1982). Two strips were cut from the midcostal

diaphragm (- 40 mg each). These strips were secured at resting length in separate baths

containing Krebs-Ringer bicarbonate solution, which was supplemented with 5 mM

glucose, insulin (1 unit/ml), 0.17 mM leucine, 0.10 mM isoleucine, and 0.20 mM valine

to improve protein balance, and 5 mM cycloheximide to inhibit protein synthesis.

Diaphragm strips were maintained at resting length by securing both ends to a solid

plexiglass support. Muscle strips were suspended vertically in the organ bath. The

medium was continuously gassed with 95% 02-5% C02, and temperature was maintained

at 37C with a recirculating water bath. After a 30-min pre-incubation period, the media

was drained, and fresh media was quickly added for a 2-hour incubation. Rates of total

protein breakdown were measured by assaying tyrosine release into the medium

according to the spectrofluorometric method of Waalkes and Udenfriend (1957).









20 S Proteasome Activity

The in vitro chymotrypsin-like activity of the 20S proteasome was measured

fluorometrically by following the release of free AMC from the synthetic substrate Suc-

Leu-Leu-Val-Tyr-AMC (Affiniti Research) using techniques described by Stein et al.

(1996). Briefly, -50 mg of midcostal diaphragm tissue was homogenized in buffer

containing 50 mM Tris base, 1 mM EDTA, 1 mM EGTA, 1 [tM Pepstatin-A, 50 [tM E-

64, and 10% glycerol. After initial centrifugations, the supernatant was collected and

centrifuged at 100,000 x g in an ultracentrifuge for 1 hour at 4oC. This supernatant

fraction was used to measure protein content using the Bradford method (Bradford 1976),

and to measure 20S proteasome activity as follows. Ten [tg of protein was reacted with

the synthetic peptide substrate for chymotrypsin-like activity (Suc-LLVY-AMC) in a

reaction mixture containing 50mM Tris-HC1, 1 mM DTT, and 5 mM MgCl2. One aliquot

from each sample was incubated with an inhibitor of the chymotrypsin-like proteasomal

activity, lactacystin (Boston Biochem), while the other was not incubated with the

inhibitor. Samples were incubated for 30 min at 370C before the addition of substrate.

The change in fluorescence was measured at an excitation wavelength of 380 nM and

emission of 460 nM. The difference between the activities of the proteasome with and

without inhibitor was used as the 20S proteasome activity.

Statistical Analysis

Comparisons between groups were made by a one-way ANOVA. Where significant

differences were found, Tukey's HSD test was implemented post hoc. Independently, the

effects of anesthesia were compared using a Student's t-test on SBS and CON group data.

Significance was established apriori at p<0.05.










Results

Systemic and Biologic Responses to Treatment

The MV protocol did not significantly change body mass for any of the 5 groups

(Table 4-1), indicating that our schedule of nutrition and rehydration was adequate. The

ratio of total costal diaphragm mass to final body mass was not significantly different

between the 5 groups (p=0.501).

There were no signs of infection in any animals, and only 1 MVS animal was

eliminated from the study due to evidence of barotrauma to the lungs on post-mortem

examination. Systolic blood pressure was maintained at 70-110 mmHg in all groups, and

arterial pH, P02, and PCO2 were maintained within physiological ranges for both MV

groups. The SB animals were mildly hypoxic, hypercapnic, and acidotic, as expected due

to the anesthesia. Body temperature was kept at 37 + 1C during the 12 hour protocol.

Effects of Anesthesia on Diaphragm Contractile Properties

The maximal tetanic force was not different between the SBS and CON groups

(25.09 + 0.41 N/cm2 vs. 25.33 + 0.50 N/cm2, respectively). Likewise, the force-frequency

curves and fatigue data were similar between these two groups (Figures 4-1, 4-2, and 4-

3), and contractile parameters did not differ (Table 4-3). Thus, 12 hours of sodium

pentobarbital anesthesia did not affect in vitro contractile properties of the diaphragm.

Effects of Mechanical Ventilation on Contractile Properties

Twelve hours of controlled MV reduced maximal tetanic force production by -17%

(21.00 + 0.71 N/cm2 vs. 25.43 + 0.50 N/cm2 in CON animals). The force-frequency curve

of the MVS group was shifted downward and to the right of the CON group (Figure 4-1).

This indicates a reduction in force generation at all stimulation frequencies tested. The

fatigue protocol produced curves of similar shape for all groups (Figure 4-2), but the









MVS group generated a significantly lower amount of force compared to CON, SBS, and

MVT groups where indicated. When the fatigue data are expressed as percent of initial

force (Figure 4-3), there are no significant differences between the 5 groups at any time

point (p= 0.230).

One-half relaxation time (/2 RT) of maximal twitch was significantly shorter in the

MVS group compared to CON, while 12 time to peak tension (/2 TPT), rate of force

development, and rate of relaxation were not different for either maximal twitch or

maximal tetanic forces (Table 4-3).

Effects of Trolox on Contractile Properties

Trolox supplementation during 12 hours of MV completely attenuated the loss of

maximal force generation. The MVT group was not significantly different from CON at

any stimulation frequency tested (Figure 4-1). Animals receiving Trolox during MV

maintained a greater force generating ability following the fatigue protocol compared to

the unsupplemented MVS group (Figure 4-2). Trolox during MV significantly prolonged

the rate of relaxation of maximal twitch compared to CON, but did not affect other

contractile parameters (Table 4-3).

Twelve hours of Trolox infusion without MV (i.e. the SBT group) greatly increased

subject mortality rate. Only three SBT animals survived the treatment. Further,

diaphragms from these animals showed impaired force production compared to CON.

The force-frequency curve of the SBT group was shifted downward and to the right of

the CON group and closely resembles the MVS group (data not shown). Trolox

supplementation to SB's also significantly reduced 12 TPT of maximal tetanic forces

compared to CON animals (Table 4-3).









Protein Degradation

Twelve hours of controlled MV significantly elevated total in vitro protein

degradation (+105%), as measured by the release of free tyrosine, compared to CON

(Figure 4-4). However, protein degradation of the MVT group (16% increase compared

to CON) was not significantly different from CON (p=0.797). There were no significant

differences in protein degradation between CON and SBS groups (p=0.351).

20S Proteasome Activity

The chymotrypsin-like activity of the 20S proteasome was significantly increased

in the MVS group compared to the CON group (+76%) (Figure 4-5). Trolox attenuated

the MV-induced increase in proteasome activity (+26% compared to CON, p=0.647).

Discussion

Major Findings

The major findings of this study are: 1) Trolox supplementation during 12 hours of

controlled MV attenuates diaphragmatic contractile dysfunction and whole muscle

proteolysis; 2) 12 hours of anesthesia and spontaneous breathing do not affect contractile

function or protein degradation within the diaphragm; 3) Proteolysis is elevated during 12

hours of MV in part due to increased chymotrypsin-like activity of the 20S proteasome,

and 4) Trolox supplementation during normal spontaneous breathing shifts redox balance

to a reductive state which actually impairs diaphragmatic function.

MV and Diaphragmatic Dysfunction

These data support our previous studies (Powers et al. 2002, Shanely et al. 2003)

that show a reduction in maximal force production with prolonged MV. These data agree

with other MV studies with rats (Le Bourdelles et al. 1994), baboons (Anzueto et al.

1997), piglets (Radell et al 2002), and rabbits (Sassoon et al. 2002, Capdevila et al.









2003). In the present study, maximal tetanic tension was decreased -17% with 12 hours

of controlled MV. Le Bourdelles et al. (1994) demonstrated that 48 hours of MV

significantly reduced diaphragmatic force without altering protein concentrations or

enzyme activities. Anzueto et al. (1997) found a decrease in transdiaphragmatic pressure

and diaphragmatic endurance after 11 days of MV in baboons. However, the use of long-

lasting neuromuscular blockers in this study may have affected diaphragm responses. We

have shown that the degree of diaphragmatic dysfunction is proportional to the length of

time of MV (Powers et al. 2002).

Radell and colleagues (2002) demonstrated that diaphragmatic dysfunction induced

by 5 days of MV in a piglet model is not associated with alterations in nerve function or

neuromuscular transmission. With respect to muscle fiber composition, Capdevila et al.

(2003) found significant atrophy of type IIa and lib fibers, with no changes in type I

fibers, in the rabbit diaphragm ventilated for 51 hours. In the rat, 4 days of MV caused a

decrease in the percentage of type I fibers, and an increase in hybrid fibers co-expressing

type I and II MHC (Yang et al. 2002). Also, Shanely and colleagues (2002) found

atrophy of all fiber types that was greatest in type II fibers after just 18 hours of MV.

Finally, significant myofibril damage in the diaphragm is evident after MV (Sassoon et

al. 2002).Together, these alterations in diaphragmatic structure may lead to the weakness

characteristic of a mechanically ventilated diaphragm muscle.

In the present study, Trolox completely attenuated the decrease in maximal specific

tension that occurs during MV. These animals were not different from CON or SBS

animals with respect to maximal tetanic tension, maximal twitch tension, force-frequency

relationships, and fatigue data.









Oxidative Stress and Trolox

Unloaded skeletal muscle is susceptible to oxidative stress during periods of disuse

(Kondo et al. 1993a, 1993b). During MV, the diaphragm muscle is both unloaded and

passively shortened (Racz et al. 2003). These stimuli are likely to stimulate the

unloading-induced atrophy typical of prolonged MV. Previous studies from our

laboratory have measured an increase in protein oxidation and lipid peroxidation with 18

hours of MV (Shanely et al. 2002), and as early as 6 hours of MV (Zergeroglu et al.

2003). In the present study, we measured a decrease in total and non-protein thiols with

12 hours of MV consistent with oxidative stress (data not shown). However, Trolox did

not prevent the loss ofthiol groups during MV. It may be that specific proteins, such as

myosin, are preferentially spared from oxidation while others are not. If true, we were not

able to detect these proteins in crude homogenate. However, we postulate that Trolox is

functioning as an antioxidant to prevent oxidative stress. Several studies have shown that

Trolox reduces oxidative stress induced by cumene hydroperoxide (Persoon-Rothert et al.

1990), methylmercury intoxication (Usuki et al. 2001), and other oxyradicals (Wu et al.

1990, Wu et al. 1991, Zeng et al. 1991, Walker et al. 1998). Wu and colleagues (1990)

have even demonstrated antioxidant actions of Trolox in three human cell types exposed

to oxyradicals. While we were unable to clearly demonstrate the prevention of oxidative

stress with Trolox, it is likely that Trolox is acting as an antioxidant to scavenge reactive

oxygen species produced in the diaphragm during 12 hours of controlled MV.

MV and Proteolysis

Twelve hours of controlled MV significantly increased (+105%) the release of

tyrosine from in vitro diaphragm strips. This agrees with a previous study from our

laboratory reporting increased tyrosine release after 18 hours of MV (Shanely et al.










2002). Tyrosine release is frequently used as an indicator of total protein degradation

(Lowell et al. 1986) since tyrosine is neither synthesized nor degraded by skeletal muscle.

In our previous 18-hour MV study, we determined that total calpain-like and 20S

proteasome activities were elevated, indicating a contribution of these two proteolytic

pathways to diaphragmatic proteolysis during MV (Shanely et al. 2002).

Importantly, Trolox attenuated the increase in total protein degradation induced by

MV (Figure 4-4). Likewise, the chymotrypsin-like activity of the 20S proteasome was

elevated during MV, but this increase was prevented with Trolox (Figure 4-5). Many

studies indicate that the proteasome is responsible for -70-80% of the increased cellular

protein degradation following an oxidative stress (Grune et al. 1995, Grune et al. 1996,

Grune and Davies 1997). More specifically, it appears the 20S proteasome is responsible

for the degradation of oxidized proteins since the 26S proteasome is inhibited/inactivated

by oxidative stress (Reinheckel et al. 1998). Recognition of exposed hydrophobic patches

is the proposed mechanism by which the proteasome selectively degrades oxidatively

modified proteins (Grune et al. 1997). Oxidative damage to a protein leads to partial

unfolding and exposure of normally shielded internal hydrophobic patches that are

recognized by the proteasome, which catalyzes the degradation of that protein.

The diaphragmatic atrophy and contractile dysfunction that occur with prolonged

MV are likely the result of increased oxidative modification of proteins, leading to

proteolytic attack and degradation. This loss of protein, especially contractile protein,

would result in atrophy and decreases in maximal force production. Clinically, this would

manifest as difficulty weaning from the ventilator.









Critique of Experimental Model

It was necessary to use an animal model due to the invasive nature of this study.

The rat was selected due to the similarities in anatomy and function of the rat and human

diaphragms. Also, controlled MV, which is not as common clinically as pressure assist

MV, was used because of the rapid onset of diaphragmatic atrophy characteristic of

controlled MV.

Prolonged anesthesia is known to lead to hypoxia, hypercapnia, and mild acidosis

due to the reduced ventilatory drive in an anesthetized animal (Powers et al. 2002). While

these effects could theoretically affect in vitro diaphragm function, our data demonstrate

that sodium pentobarbital anesthesia does not impact skeletal muscle function in vitro. A

comparison of the CON and SBS data indicate no significant differences in force

generation, total proteolysis, or proteasome activity. Thus, the diaphragmatic dysfunction

and proteolysis induced by MV were not caused by prolonged sodium pentobarbital use.

We attempted to include a group to control for Trolox during spontaneous

breathing (SBT group). However, only 3 animals from this group survived the entire 12

hours, and these were not included in statistical analyses. It is not surprising that Trolox

was harmful in these animals that were not exposed to oxidants because Trolox is a

strong reductant. We hypothesize that Trolox shifted the redox balance to a reductive

state in the spontaneously breathing animals that impaired diaphragmatic function.

Conclusions

Our results support earlier conclusions that short-term controlled MV leads to

diaphragmatic contractile dysfunction and increased protein degradation. Our data clearly

demonstrate that an antioxidant, Trolox, effectively prevented contractile impairments

and proteolysis during MV. Oxidative damage and atrophy are implicated in MV-induced









contractile deficits. Oxidative damage to proteins during MV likely increases proteolytic

degradation, which would contribute to diaphragmatic weakness. Trolox effectively

spares the unloaded diaphragm from contractile dysfunction, oxidative stress, and protein

degradation during 12 hours of controlled MV. However, it is not warranted during

normal spontaneous breathing, and can actually cause contractile dysfunction under such

conditions.

Future studies aiming to prevent force losses, oxidative stress, and protein

degradation during MV might examine another vehicle and route of Trolox

administration. In addition, other antioxidants need to be tested. The use of an antioxidant

such as Trolox may prove beneficial in the clinical setting where weaning difficulties are

encountered due to diaphragmatic atrophy and weakness. This is a serious clinical issue

that warrants further investigation.















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BIOGRAPHICAL SKETCH

Jenna Betters was born in New Smyrna Beach, Florida, and graduated salutatorian

of her high school class in 1997. She received her bachelor's degree in biology from the

University of North Florida, Jacksonville, Florida, in May of 2001, where she was a

member of Phi Kappa Phi Honor Society, University Scholar's Honor Society, and

Golden Key International Honor Society. She began a master's program in exercise

physiology at the University of Florida, Gainesville, Florida. She has worked for two

years as a fitness trainer, and three years as a research assistant in the Molecular

Physiology Laboratory within the Center for Exercise Science. Jenna has been accepted

into the doctoral program and will pursue a Ph.D. in exercise physiology from the

University of Florida.