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Mechanisms of Mechanical Ventilation-Induced Oxidative Stress in the Diaphragm

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MECHANISMS OF MECHANICAL VENTILATION-INDUCED OXIDATIVE STRESS IN THE DIAPHRAGM By DARIN VAN GAMMEREN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Darin Van Gammeren

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This dissertation is dedicated to my parents, Bill and Carol.

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iv ACKNOWLEDGMENTS First and foremost I would like to tha nk my mentor, Dr. Scott Powers, for his guidance. Without his tutelage, this proj ect would not have been possible and my education surely lacking. I would also like to thank my committee members, Dr. David Criswell, Dr. Stephen Dodd, and Dr. Glenn Walter for their invaluable input. Further, a big thanks goes to “team diaphragm” (D arin Falk, Melissa Deering, and Dr. Keith DeRuisseau) for the camaraderie and incalculable assistance on this project. Finally, I would like to thank Joel French for his technical assistance.

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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 2 LITERATURE REVIEW.............................................................................................4 Mechanical Ventilation-Induced Diaphragmatic Dysfunction.....................................4 Muscle Force.........................................................................................................4 MV-Induced Atrophy............................................................................................6 Structural Injury.....................................................................................................6 Oxidative Stress.....................................................................................................8 Mitochondria..................................................................................................9 Reactive iron................................................................................................10 Xanthine oxidase..........................................................................................10 NADPH oxidase...........................................................................................11 Nitric oxide (NO).........................................................................................11 Summary.....................................................................................................................13 3 METHODS.................................................................................................................15 Animals and Experimental Design.............................................................................15 Experimental Protocol................................................................................................15 Diaphragmatic Measurements....................................................................................17 Contractile Measurements...................................................................................17 Biochemical Measurements.................................................................................18 NADPH oxidase activity..............................................................................18 Myeloperoxidase (MPO) activity.................................................................18 Glutathione...................................................................................................19 Protein carbonyls..........................................................................................19 Nitric oxide synthase (NOS)........................................................................19

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vi 3-nitrotyrosine..............................................................................................21 Statistical Analysis......................................................................................................22 4 RESULTS...................................................................................................................23 Systemic and Biologic Response to MV....................................................................23 Contractile Dysfunction..............................................................................................23 NADPH Oxidase and MPO Activity..........................................................................24 Glutathione.................................................................................................................24 Protein Carbonyls.......................................................................................................25 Nitric Oxide Synthase (NOS).....................................................................................25 3-Nitrotyrosine............................................................................................................25 5 DISCUSSION.............................................................................................................33 Overview of Principal Findings..................................................................................33 Role of NADPH Oxidase in MV-induced Oxidative Stress.......................................33 Role of Nitric Oxide Synthase (NOS) in MV-induced Oxidative Stress...................36 Critique of Experimental Model.................................................................................37 Conclusions.................................................................................................................38 Future Directions........................................................................................................38 LIST OF REFERENCES...................................................................................................40 BIOGRAPHICAL SKETCH.............................................................................................46

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vii LIST OF TABLES Table page 1 Arteriole blood values duri ng SB and MV protocol................................................26 2 NADPH oxidase and myeloperoxidase (MPO) activity in the diaphragm..............26 3 Nitric oxide synthase (NOS) pr otein levels in the diaphragm.................................26 4 Nitrate and nitrite leve ls in the diaphragm...............................................................26 6 Diaphragmatic levels of 3-nitrotyrosine within insoluble proteins..........................27 7 Diaphragmatic protein levels of cy tosolic (soluble) 3-nitrotyrosine........................27 8 Diaphragmatic mitochondrial prot ein levels of 3-nitrotyrosine...............................27 9 Diaphragmatic protein levels of 3-nitr otyrosine within membrane proteins...........27

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viii LIST OF FIGURES Figure page 1 Effect of 18 hours of mechanical ven tilation (MV) on the diaphragmatic forcefrequency response...................................................................................................28 2 Effect of 18 hours of mechanical ven tilation on the diaphr agmatic levels of reduced glutathione..................................................................................................29 3 Effect of 18 hours of mechanical ven tilation on the diaphr agmatic levels of protein carbonyls......................................................................................................30 4 Representative western blots illustrati ng the diaphragmatic protein levels of NOS..........................................................................................................................31 5 Representative western blots illustrati ng the diaphragmatic protein level of 3nitrotyrosine.............................................................................................................32

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ix Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MECHANISMS OF MECHANICAL VENTILATION-INDUCED OXIDATIVE STRESS IN THE DIAPHRAGM By Darin Van Gammeren August 2005 Chair: Dr. Scott Powers Major Department: Applied Physiology and Kinesiology Mechanical ventilation (MV) is associat ed with oxidative stress and contractile dysfunction in the diaphragm. The pathways responsible for the production of oxidants in the diaphragm during MV remain unknown. To address this issue, these experiments tested the following hypotheses: 1) NADPH oxidase activity is increased during MV and contributes to the oxidative stress and contractile dysfuncti on of the diaphragm; and 2) diaphragmatic nitric oxide synthase (NOS) le vels are elevated in the diaphragm during MV and contribute to nitration of proteins in the diaphragm. To test these postulates, rats were mechanically ventilated for 18 hours w ith a subset of animals receiving the NADPH oxidase inhibitor, apocynin (4 mg/kg body weight). Diaphragmatic NADPH oxidase and NOS activities were measured along with prot ein levels of all three NOS isoforms. Further, 3-nitrotyrosine levels in the diaphragm were measur ed as an index of protein nitration. Compared to control, MV result ed in diaphragmatic oxidative stress and a significant decrease (-10%) in the maximal speci fic force of the diaphragm. MV did not

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x increase diaphragmatic NADPH oxidase ac tivity above control. Nonetheless, the administration of apocynin attenuated MV-indu ced contractile dysfunction. Interestingly, treatment with apocynin did not diminish diaphragmatic NADPH oxidase activity but protected the diaphragm against MV-induced oxidative stress. Moreover, MV did not promote an increase in diaphragmatic protei n levels of eNOS, nNOS, or iNOS or NOS activity. Consistent with these findings, MV did not elevate diaphragmatic protein levels of 3-nitrotyrosine in any region of the di aphragm including the insoluble, cytosolic, mitochondrial, and membrane protein fracti ons. Therefore, we conclude that MVinduced oxidative stress in the diaphragm is not due to increases in NADPH oxidase activity or increased NO production by NOS. More over, our results suggest that apocynin attenuates the MV-induc ed diaphragm contractile dysfunc tion and oxidative stress via its antioxidant properties, not through th e inhibition of NADPH oxidase.

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1 CHAPTER 1 INTRODUCTION Mechanical ventilation (MV) is used to ma intain adequate alveolar ventilation in patients incapable of doing so on their own. The removal of patients from the ventilator is termed weaning. Difficulty in weaning is often defined as a weaning procedure requiring more than 48 hours to permanently re move patients from the ventilator (1). Importantly, difficulties in weaning occur in approximately 25% of patients utilizing MV (1). Clinically, this is significant becaus e weaning accounts for grea ter than 40% of the total time on the ventilator (2). There are two major modes of MV: pressu re-assist and controlled. As the name implies, pressure-assist MV assists the patients’ inspiratory efforts, while during controlled MV, the ven tilator delivers all of these br eaths, therefore rendering the inspiratory muscles inactive. Pressure-assist MV is commonly utilized in adult patients suffering from acute respiratory failure whereas controlled MV is u tilized in instances where patients suffer from a spinal cord in jury or during surgery (3). Furthermore, controlled MV is commonly used in pediatric situations (3). Given that controlled MV completely inactivates the respiratory musc les, as revealed by the absence of EMG activity in the diaphragm during MV, it is likely that diaphragmatic atrophy and dysfunction associated with MV occur ra pidly using this mode of MV (3-5). To study the effects of MV on the diaphrag m, an animal model must be utilized because of the invasive nature of removing diaphragm muscle samples. Thus far, four animal models have been utilized including the baboon, pig, rat, a nd rabbit. Of these

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2 models, the rat has been util ized in our laboratory due to similarities to the human diaphragm in fiber type, biochemical propert ies, anatomical features, and physiological function (6-8). Previously, our laboratory has demonstrated a decrement in the specific force of the diaphragm following as little as 12 hours of MV (4). Further, this deficit is exacerbated to a 60% loss in force following 48 hours of MV (9). During MV, our laboratory (10) and others (9, 11-14) have not ed an increase in diaphrag matic atrophy; however, since the specific force of the diaphragm is normalized per cross sectional area, the decline in muscle force production is not due to atrophy alone. One proposed mechanism by which diaphr agmatic dysfunction may occur during MV is via oxidative stress-induced injury to the diaphragm. Our group has found an increase in protein oxidation a nd lipid peroxidation following as little as six hours of MV (10, 15). These oxidized proteins can be targ eted by the proteasome proteolytic system where they are degraded by the 20S proteasom e, thereby accelerating muscle atrophy (16, 17). Further, an increase in oxidative stress can damage proteins involved in excitationcontraction coupling, reducing muscle for ce production (18-20). The physiological significance of MV-induced oxida nt stress in the diaphr agm has been confirmed by recent experiments demonstrating that infusi on of the antioxidant, Trolox, prevents MVinduced contractile dysfuncti on in the diaphragm (21). Various sources of reactive oxygen speci es (ROS) exist in skeletal muscle including the calcium-activated enzyme, NADPH oxidase. This source of ROS is responsible for the one electron reduction of oxygen into superoxide using NADPH or NADH as the electron donor (22). The inhib ition of NADPH oxidase derived superoxide

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3 can be achieved with the addition of apocynin (4-hydroxy-methoxyacetophenone; acetovanillone). Apocynin most likely re duces superoxide formation by blocking sulfhydryl groups and inhibiting NADPH oxidase enzyme assembly (23). Previous investigators have successfully used apocynin in vitro and in vivo to inhibit NADPH oxidase activity in skeletal muscle (22, 24) NADPH oxidase has been well characterized in the mammalian diaphragm and remains as a possible source for oxidant production in inactive skeletal muscle. Also, nitric oxide (NO) has been reported to be produced in immobilized locomotor muscle and therefore could al so contribute to oxidative inju ry in the diaphragm during MV (25). NO is produced by calcium-activated cellular enzyme, nitr ic oxide synthase (NOS). NO can react with superoxide to form th e highly reactive peroxynitrite molecule that can nitrosylate proteins in skeletal muscle. Given that MV-induced oxidant stress is associated with diaphragmatic contractile dysfunction, determining which oxi dant producing pathways are responsible for the generation of reactive oxygen species is important. Based on preliminary experiments, we formed the working hypothesis that MV results in an increase in oxidant production in the diaphragm via increased NADPH oxidase activity and an increase in nitric oxide synthase (NOS). More specifically, the curren t experiments are designed to test two hypotheses: 1) NADPH oxidase activity is incr eased in the diaphragm during MV and contributes to diaphragmatic oxidative injury and contractile dysfunction; and 2) diaphragmatic NOS levels are elevated in the diaphragm during MV and contributes to the nitration of protei ns in the diaphragm.

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4 CHAPTER 2 LITERATURE REVIEW Mechanical ventilation (MV) is utilized to maintain adequate alve olar ventilation in patients incapable of doing so on their own. The process of removing patients from the ventilator is termed weaning, and problems in weaning occur in approximately 25% of patients exposed to MV for two or more days (1). One of the proposed mechanisms by which weaning difficulties occur is due to impairments in diaphragmatic strength and endurance (4, 9, 11-13, 26-29). Importantly, rece nt experiments in our laboratory suggest that MV-induced oxidative stress contribu tes to both diaphragmatic atrophy and contractile dysfunction (10, 15). Indeed, it is well establ ished that an increase in oxidative modification of proteins and lipid s in the diaphragm can promote skeletal muscle atrophy and dysfunction (10, 15). This review will outline our current understanding of MV-induced diaphragm dys function and will also provide a brief overview of oxidant producing pathways th at could be responsible for MV-induced oxidative damage in the diaphragm. Mechanical Ventilation-Induced Diaphragmatic Dysfunction Muscle Force Utilizing a variety of different animal models, diaphragmatic dysfunction has been evaluated following MV (4, 9-1 2, 14, 15, 21, 26-28, 30-35). These experiments reveal that the in vivo transdiaphragmatic pressure of baboons, piglets, and rabbits is significantly reduced following MV (11, 27, 28). For example, diaphragmatic force decrements have been shown to occur as earl y as one day in rabbits and three days in

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5 piglets; these force decrements are exacer bated with time on the ventilator (27, 28). Further, there is a 40-50% decline in the pr essure-generating capaci ty of the diaphragm after 3 days in rabbits, 5 days in piglets, and 11 days in baboons (11, 27, 28). Also, it has been shown that following a prolonged period of MV, animals cannot sustain diaphragmatic force under an inspiratory re sistive load, indicative of an endurance decrement of the diaphragm (11). Importa ntly, these impairments in diaphragmatic function are not attributable to changes in lung volume and abdominal compliance or the function of the phrenic nerve a nd neuromuscular junction (11, 27). In addition to the aforementioned in vivo experiments, in vitro preparations have also been utilized to determine the force of isolated rat and ra bbit diaphragm strips removed from animals following varying peri ods of MV. A 30-50% reduction in the maximal isometric specific force of the diaphr agm occurs after one to three days of MV (4, 9, 12, 13, 28) with as little as 12 hours of MV resulting in an approximately 20% decrement in force (4, 26). The decrements in force cannot be attributed to atrophy alone due to the fact that the force of the diaphrag m in these studies has been normalized to the cross-sectional area. Furthermore, the dia phragm strip is set at the optimal length; therefore, the force decrement cannot be due to altered muscle operating length. Finally, anesthetic agents and neuromuscular blocke rs are not the cause of the diaphragmatic dysfunction following MV. This has been experimentally demonstrated by omitting neuromuscular blockers from se veral studies and the anesthetic agent has been controlled by utilizing a spontaneously breathing group of animal s under anesthesia, but not undergoing MV (36).

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6 The endurance of the diaphragm follo wing MV has also been assessed in vitro (13, 28, 32, 33). Conclusions on the fatigue re sistance of these diaphragm strips are inconsistence with studies repor ting an increase (32), decreas e (33), or no change in the endurance of the diaphragm following MV (13, 28) Therefore, it is not clear what the effects of MV are on the endurance of the diaphragm. In humans, it is much more difficult to study the effects of MV on diaphragmatic function. Confounding factors such as vari ous disease states, drugs, and modes of ventilation make it difficult to determine the cause of respiratory muscle dysfunction. Further, assessment of diaphragm function in humans is complicated given that the obtainment of diaphragm samples is invasive However, histopathologic analysis of diaphragms from 13 neonates ventilated 12 da ys or more suggests that diaphragm fiber atrophy occurs (37). Moreover, 33 patients with various diseas es exposed to two or more days of MV exhibited a 50% decrement in the twitch transdiaphragmatic pressure from supramaximal magnetic stimulation of the phrenic nerve when compared to normal subjects (29). Therefore, although data fr om human studies are limited, the current results are consistent with animal experime nts indicating that pr olonged MV results in diaphragmatic atrophy and contractile dysfunction. MV-Induced Atrophy MV-induced diaphragm atrophy has been report ed in almost all animal experiments (9, 11-14). MV-induced diaphragmatic atrophy occurs more rapidly (e.g., 18 hours) than disuse atrophy of peripheral skeletal musc les (9, 10, 12). Disuse muscle atrophy can occur due to a decrease in prot ein synthesis (38), an increase in protein degradation (39), or a combination of both. Our laboratory has shown a decrease in protein synthesis during as little as six hours of MV (34). Moreover, mRNA fo r insulin-like growth factor

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7 (IGF-1) and type I and IIx myosin heavy chai n are depressed after 18 to 24 hours of MV (13, 34). Importantly, our group has reported an increase in proteolysis after 18 hours of MV (10). Based upon the relativ ely long half-life of many skel etal muscle proteins, it is feasible that the rapid onset of diaphragmatic atrophy is prim arily due to a rapid onset of proteolysis. There are three primary pathwa ys involved in proteolysis in skeletal muscles: 1) lysosomal proteases (cathepsins), 2) calcium ac tivated neutral proteases (calpain), and 3) the proteasome. In regard to proteas e activation in the diaphragm during MV, Shanely et al. (10) have reported a greater than twofol d increase in diaphragmatic calpain activity and approximately a fivefold increase in 20S proteasome activity following 18 hours of MV. Calpains are responsible for the rel ease of myofilaments from the sarcomere and allow them to be degraded by the proteasome (40). The proteasome system consists of the ATP-dependent 26S proteasome, which requires the ubiquitination of proteins prior to degradation and the 20S proteasome that de grades proteins oxid ized by reactive oxygen species (ROS) without the need for ATP or ubiquitin (36). An increase in calpain-like activity and 20S proteasome activity has been reported in the diaphragm following MV (10). Structural Injury During MV, alterations in the structure of the diaphragm such as disrupted myofibrils (14, 28), an increased number of lipid vacuoles in the sarcoplasm and abnormally small mitochondria with focal memb rane disruptions have been reported in rabbits (14). Investigation on the external in tercostals revealed si milar findings (14). In contrast, inactive hindlimb muscles removed from anim als undergoing MV do not exhibit these modifications (28). Myofibril disrupti on is physiologically significant because an

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8 increase in abnormal myofibrils has been s hown to be significan tly correlated with decrements in diaphragmatic force output (28). A definitive explanation for MV-induced myofilament disruption does not currently exist. However, at least two possible explanations exist. First, the calcium-activated protease calpain could be releasing the myofilaments from the sarcomere (10). Secondly, periods of spontaneous breathing could occur during MV that w ould reload the diaphragm. Reloading of hindlimb muscles following prolonged periods of disuse has been associated with an increased susceptibility to muscle fiber injury (41). Nonetheless, we have demonstrated that two hours of reloading the diaphragm followi ng 24 hours of MV does not exacerbate MV induced contractile dysfunction or cause me mbrane damage (35). Therefore, during periods of 24 hours of MV or less, spontane ous breathing does not appear to contribute to the structural damage that occurs in the diaphragm. Oxidative Stress It is well established that when cellular oxidant production exceed s the capacity of intracellular antioxidants to scavenge thes e oxidants, oxidative damage to cellular biomolecules occurs. In this regard, our research team has reported an increase in protein oxidation and lipid peroxidation in the diaphragm following various periods of controlled MV (10, 15). Interestingly, MV-induced oxidativ e injury occurs in the diaphragm within as few as six hours after the onset of MV (15). In this study, oxidized proteins were separated using SDS-PAGE and it was determined that the contractile proteins, actin and myosin, are oxidized in the dia phragm during prolonged MV (15). At present, the pathways responsible for MV-induced oxidative stress in the diaphragm are unknown. Major ox idant producing pathways in cells include the electron

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9 transport chain in the mitochondria, reactive iron, xanthine oxidase, NADPH oxidase, and nitric oxide synthase. A brief overview of each of these pathways follows. Mitochondria Mitochondria primarily func tion to produce ATP; however they have also been shown to produce superoxide radicals (42). The major site of superoxide production within the mitochondri a is the electron transport chain (42). Early components of the electron transport chain can leak electrons directly onto oxygen while most of the electrons are transferred to th e next component of the chai n. It is this leakage of electrons that generate s superoxide (42). During resting conditions (state four re spiration), there is a high degree of reduction of the electron carrier s and a limited supply of ADP as compared to periods of increased muscle contractile activity (state thr ee respiration) (43). Therefore, during state four respiration, there is a greater proportional amount of superoxide production and the production of superoxide has been estimated to account for more than two percent of the oxygen consumed (43). Moreover, with increasing concentrations of oxygen, there is an increase in electron leakage and, therefore, superoxide production (44). Howe ver, at physiological concentrations of oxygen, it has been estimated that only one to three percent of the oxygen reduced in the mitochondri a forms superoxide (44). Th e low rate of leakage is most likely due to low oxygen concentrations within the mitochondria and the facilitation of electron flow by electron carrier comple xes. At present, it is unknown if the mitochondria are a significant source of ROS in the diaphragm during MV.

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10 Reactive iron The highly reactive hydroxyl radical can be produced from the reaction of superoxide with hydrogen peroxi de, but without reactive metals present, this reaction is too slow to be of physiologica l significance (45). In contrast, in the presence of a metal catalyst like iron or copper, this reaction, te rmed the Haber-Weiss reaction, will proceed much more rapidly (45). During disuse atrophy, muscle fiber volum e decreases rapidly and cell structure changes greatly which can disturb the balance of metals (46, 47). An increase in iron has been noted throughout 12 days of muscle disuse atrophy as revealed by an increase in the microsomal fraction of iron (46, 48). Furthe r, an increase in a 54 kDa iron binding protein in the sarcoplasmic reticulum has been observed as early as four days after the initiation of skeletal muscle immobilization (46). Finally, when animals were treated with the iron chelator, deferoxamine, duri ng skeletal muscle immobilization, there was a decrease in disuse-induced lipid peroxidation and oxidized glutathione (GSSG) in the immobilized muscles (46). However, when the deferoxamine was saturated with iron, markers of oxidative st ress did not change. One of the potential sources of free iron is heme oxygenase (HO). HO degrades heme into iron, carbon monoxide, and biliverdin (49). Of the two isoforms of HO (e.g., HO-1 and HO-2), HO-1 is the inducible isoform. Increases in oxidant stress have been shown to induce HO-1 and HO can serve as a pro or antioxidant ( 49). By degrading heme and releasing iron, HO act s as a pro-oxidant. In cont rast, HO can also act as an antioxidant. For example, HO produces biliverdi n that can be converted into bilirubin, and both compounds are antioxidants. Furt her, HO produces the iron binding protein ferritin that acts as an anti oxidant by binding free iron and th erefore preventing the iron-

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11 mediated catalyzed forma tion of the hydroxyl radical (49). At present, it is unclear if HO acts as a pro-oxidant or an an tioxidant in the diaphragm and this remains as an important area for future research. Xanthine oxidase Xanthine oxidase (XO) is present in the cy toplasm of skeletal muscle and exists in two forms: NAD-dependent (type D) and supe roxide-producing (type O) (50). Both types have been shown to increase duri ng 12 days of disuse muscle atrophy with increases in type O being gr eater (2.3-fold higher). Furt her, the substrates of XO, xanthine and hypoxanthine, ar e increased during muscle disuse as well as their product (i.e., urate). The larger increase in type O XO increased the ratio of type O XO to total XO which is indicative of the conversion of t ype D to type O XO (50) It is known that this conversion is catalyzed by calcium activated neutral proteases (cal pain) (51). This is consistent with data indicating an increase in intracellular calcium during disuse atrophy (48, 52). Nonetheless, it is unclear if XOinduced production of oxi dants is operative in the diaphragm during prolonged MV. NADPH oxidase NADPH oxidase is a membrane-associated enzyme that produces superoxide via a one-electron reduction of oxygen using NAD PH or NADH as the electron donor. The enzyme exists in phagocytes (53) and nonphagocytes. Several differences exist between NADPH oxidases found in phagocytic and nonphagocytic cells including enzyme orientation, direction of superoxide production, subunit structure, and substrate preference (54). The phagocytic NADPH oxidase consists of five subunits including a plasma membrane-spanning cytochrome b558 that is composed of the p22phox and gp91phox subunits. Also, there are three subunits located in the cy tosol including the

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12 p47phox, p67phox, and p40phox. The cytosolic subunits are not associated with the membrane bound cytochrome until activation. Upon activation, the utilization of intracellular NADPH or NADH causes the tr ansfer of electrons to oxygen in the extracellular space, producing super oxide outside of the cell (22). Recently, it was discovered by Javes ghani et al. (22) that the p22phox, gp91phox, p67phox, and p47phox subunits’ mRNA and protein we re located in skeletal muscle while p40phox was only in the blood vessels. Fu rthermore, this group discovered that the four subunits are constitutively expressed in the membrane, unlike the phagocytic NADPH oxidase. Importantly, they demonstr ated that NADPH oxidase was capable of significant level of superoxide production in skeletal muscle. Given that NADPH oxidase can be activated by in creases in intracellular calcium levels, we postulate that NADPH oxidase is a possible source of oxidant production in the diaphragm during prolonged MV.Nitric oxide (NO) Nitric oxide (NO) The free radical, nitric oxide (NO), is produced by the enzyme NO synthase (NOS). There are three isoforms of NOS: 1) type I or neuronal (nNOS), 2) type II or inducible (iNOS), and 3) type III or endot helial (eNOS). All forms of NOS produce NO from L-arginine and requir e oxygen and NADPH as substrates while citrulline is a byproduct (55). The cofactors involved in this reaction include FAD, FMN, tetrahydrobiopterin, heme, and calmodulin. The binding of NOS and calmodulin is calcium dependent with only the constitutive isoforms (nNOS and eNOS) being calcium sensitive (55). The calcium sensitive binding of calmodulin is the primary regulator of the production of NO via the constitutive isof orms of NOS; however iNOS is tightly

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13 bound to calmodulin and not calcium regulated. iNOS is primarily regulated at the transcriptional level and is upregulated due to an inflammatory challenge (55). Targets of NO can be generalized into th ree main categories. First, NO can react with oxygen and superoxide to form low mo lecular weight NO derivatives such as peroxynitrite. These molecules maintain redox ac tivity and can particip ate in the transfer of electrons (56). The NO de rivative, peroxynitrite, is one of the most reactive free radicals involved in oxidative damage within skeletal muscle (57). Second, derivatives of NO can react with transition metals such as iron to produce NO-metal adducts. This is a mechanism by which NO modulates metalloprot ein function. Third, one of the main targets of NO is reduced thiols. NO can cause the S-nitrosylation of protein thiols, which is reversible (55). The formation of 3-nitr otyrosine is one of the most commonly studied covalent modifications of proteins by NO (58). Summary Previous work has demonstrated th at control MV renders the diaphragm completely inactive. During th is period of disuse, diaphragmatic force decrements occur within as little as 12 hours of MV. Further, diaphrag matic atrophy occurs but cannot account for all of the dysfuncti on due to the fact that the force of the diaphragm is normalized per cross sectional area. MV has also been associated with an increase in oxidative stress in the diaphragm. Specifically, following as fe w as six hours of MV, increased lipid peroxidation and protein oxidation occurs within the diaphr agm. This is physiologically significant because MV-induced oxidative stress can accelerate diaphragmatic proteolysis and contractile dysfunction. Numerous biochemical pathways could be involved in the increase in oxidants that cause damage to the diaphragm including the mitochondria, free

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14 (reactive) iron, xanthine oxidase, NADPH oxidase, a nd NO. While all of these pathways are potential contributors to the increase in oxidative stress that occurs during MV, the primary aim of this investigation is to determine if the NADPH oxidase pathway and NO pathway are involved in MV-indu ced oxidative injury in the diaphragm. Our long-term objective is to determine which ROS pathways are involved in oxidant production in the diaphragm during prolonged MV and to devel op specific antioxidant countermeasures to protect the diaphragm against th e detrimental effects of MV.

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15 CHAPTER 3 METHODS Animals and Experimental Design These experiments were approved by the Un iversity of Florida Animal Care and Use Committee and followed the guidelines for animal experiments set forth by the National Institutes of Health. Female, Sprague-Dawley rats (4-months old) were randomly assigned to one of five groups, n = 8 per group: 1) acute control, 2) 18 hour spontaneously breathing c ontrol (SB), 3) 18 hour SB control with NADPH oxidase inhibition (SBA), 4) 18 hour mechanically ve ntilated (MV), 5) 18 hour MV with NADPH oxidase inhibition (MVA). Experimental Protocol Animals were anesthetized with sodi um pentobarbital (60 mg/kg body weight, intraperitoneal (IP)). After reaching a surgical plane of anesthesia, the acute control animals were sacrificed immediately while the SB and MV animals were tracheostomized utilizing asep tic techniques. The SB animals breathed spontaneously for the 18-hour duration while the MV animals were mechanically ventilated with a volumedriven ventilator (Inspira, Harvard Apparatu s, Cambridge, MA) for the same duration. The tidal volume was set at approximat ely 0.55 ml/100 grams body weight with a respiratory rate of 80 breaths per minute and a positive end-expiratory pressure (PEEP) of one centimeter water. In the SBA and MVA animals, the NADPH oxidase inhibitor, apocynin, was dissolved in saline and admini stered via an IP injection prior to the

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16 experimental protocol (4 mg/ kg body weight). This dosage has been previously utilized in vivo to inhibit NADPH oxidase activity in skeletal muscle (24). The carotid artery was cannulated to permit measurement of arterial blood pressure and the collection of blood. During th e first hour of the e xperimental protocol, after approximately 9 hours and during the fi nal hour of the experiment, blood samples were analyzed for the partial pressures of O2 and CO2, arterial pH, sodium (Na+), potassium (K+), calcium (Ca++), glucose and lactate us ing an electronic blood gas analyzer (GEM Premier 3000; Instrumenta tion Laboratory, Lexington, MA). Arterial PO2 was maintained throughout the experiment by gradually increasing the FIO2 using a hyperoxic gas (range 22-25% oxygen) in both SB and MV animals. Moreover, the jugular vein was cannulated for the infusi on of saline and sodium pentobarbital (~10 mg/kg body weight/hour). Apocynin was administ ered via an IP injection (4 mg/kg body weight). Furthermore, animals received an intr amuscular injection of glycopyrrolate (0.04 mg/kg body weight) every two hours to reduce ai rway secretions. Body temperature was maintained at approximately 37oC and heart rate was monitored via a lead II electrocardiograph. Continuous care duri ng the experimental protocol included lubricating the eyes, expressing the bladde r, removing airway mucus and rotating the animal and limbs of the animal. Enteral nutrition was provided via the AIN-76 rodent diet with a nutrient composition of 15% proteins, 35% lipids, 50% carbohydrates, and vitamins and minerals (Research Diets Inc ., New Brunswick, NJ). Our planned feeding schedule was designed to provide an isocaloric diet with the nutrients administered every

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17 two hours with a gastric tube; the total administration of 69 ml is equivalent to 69 kcal/day. Following the experimental protocol, th e diaphragm was quickly removed and placed in a dissecting chamber containing Kr ebs-Hensleit saline aerated with 95/5% O2/CO2. One segment of the costal diaphragm was used to assess the in vitro contractile function of the diaphragm wh ile the remaining costal diaphragm was dissected into multiple segments (~ 50 mg) and quickly frozen in liquid nitrogen and stored at –80oC for subsequent assay. Finally, due to the negative effect of sepsis on the diaphragm, blood samples from each animal were cultured to determine if gram positive and gram negative bacteria were present in the blood. Diaphragmatic Measurements Contractile Measurements The force-frequency response of a strip of costal diaphragm was performed and normalized to the cross-sectional area (CSA) as described previously (4). Briefly, a strip of diaphragm muscle was obtained from th e midcostal region including the tendinous attachments at the central tendon and rib cage. The strip was vertically suspended in a jacked tissue bath between two plexiglas clamps with one end connected to an isometric force transducer (model FT-03, Grass Instrume nts, Quincy, MA). The tissue bath was filled with Krebs-Hensleit saline (pH = 7.4) aerated with 95/5% O2/CO2. Following a 15-minute equilibration period at 25oC, the muscle strip was stimulated with platinum electrodes surr ounding the muscle strip. A supramaximal stimulation voltage (~150%) was utilized to determine optimal contractile length (Lo) of the muscle strip by systematically adjusting the length of the muscle while stimulating it with single twitches. All contractile measurements were made at Lo.

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18 To determine the force-frequency response, each muscle strip was stimulated with 120 V pulses at 15-160 Hz with a train durati on of 500 ms. Contracti ons were separated by a two minute recovery period. Following th is protocol, the musc le strip length was measured at Lo using a caliper. The strip was then trimmed from the supporting rib and all connective tissue and fat wa s removed. The remaining strip of muscle was weighed and the CSA was determined by using the following formula: total muscle CSA (mm2) = [muscle mass/(fiber length x 1. 056)]. The density of muscle in g/cm3 is 1.056 (59). Biochemical Measurements NADPH oxidase activity The activity of NADPH oxida se was measured using a lucigenin technique as described by Cui and Douglas (60). Br iefly, muscle was homogenized in a 20 mM potassium phosphate buffer (KPO4) (pH = 7.2) containing 1 mM EGTA. One hundred microliters of supernatant was added to 900 ul of buffer (50 mM KPO4, 1 mM EGTA, 150 mM sucrose, 230 uM lucigenin, and 500 uM NADH). The change in luminescence was measured over a 10-minute period and nor malized to protein. Protein concentration of the homogenate was determined using the Bradford technique (61). Myeloperoxidase (MPO) activity MPO has been shown to be highly corre lated with the number of neutrophils present in the tissue and therefore MPO activit y was determined as an indication of the level of neutrophil infiltration into the di aphragm during our expe riments (62). MPO activity was measured using the method of S eekamp et al. (62). Briefly, muscle was homogenized in a 50 mM KPO4 buffer containing 0.5% HTAB and 5 mM EDTA (pH = 6.0). Homogenate was sonicated for 10 sec onds and centrifuged cold at 3000 x g for 30 minutes. Ten microliters of homogenate was added to 290 ul of the reaction solution (50

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19 mM KPO4 buffer, 3% H2O2, 1% o-dianisidine, pH = 6.0). The change in absorbance was measured for three minutes and the value wa s expressed as activity units per gram of muscle (wet weight). Glutathione As a marker of oxidative stress, reduced glutathione levels were measured in a segment of costal diaphragm using a commerc ially available kit (Cayman Chemical, Ann Arbor, MI). The principal of this assay is that the sulfhydryl group of glutathione (GSH) reacts with 5,5-dithiobis-2-ni trobenzoic acid (DTNB) to produce a yellow-colored 5-thio2-nitrobenzoic acid (TNB). The mixed disulf ide that is produced by this reaction is reduced by glutathione reductase to recycle the GSH and produce more TNB. The rate of TNB production is directly proportional to the recycling reaction and, therefore, reflective of the concentration of GSH in the sample. Reduced GSH was calculated by subtracting the amount of oxidized glutat hione (GSSG) from the amount of total glutathione. The reduced form of GSH is the most prevalent in biological systems. Protein carbonyls The carbonyl assay is a general assay of oxidative damage to proteins. The principle of the assay is that several reactive oxygen species attack amino acid residues in proteins to produce products with carbonyl gr oups which can be measured after reaction with 2,4-dinitrophenylhydrazine. To measure protein carbonyl levels in our muscle samples, a segment of costal diaphragm was homogenized in phosphate buffered saline (pH = 7.4) and centrifuged at 1000 x g for 30 mi nutes. The supernatant was adjusted to contain two mg protein/ml and reacted w ith 2,4-dinitrophenylhydrazine overnight. Protein carbonyl levels in each sample were then detected via a commercially available ELISA kit (Zenith Technology Corporation, Dunedin, NZ).

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20 Nitric oxide synthase (NOS) Protein levels of all three isoforms of NOS (eNOS, n NOS, iNOS) were detected by Western analysis. Crude muscle homogenate (75 ug protein) wa s loaded onto a 7.5% tris-glycine SDS polyacrylamide gel and sepa rated via electrophoresis (100 V, 1.5 hours). Proteins were then transferred to nitroce llulose (2 hours at 275 mA) and the membrane was blocked in 5% non-fat dry milk. The membrane was then exposed to a monoclonal antibody for eNOS, nNOS, and iNOS at a dilution of 1:500 (BD Transduction Laboratories, Lexington, KY) followed by exposure to a HRP conjugated anti-mouse secondary antibody. Positive controls were included on each gel including human endothelial cell lysate, rat cerebrum lysa te, and mouse macrophage stimulated with IFN /LPS for eNOS, nNOS, and iNOS, respective ly. Further, membranes were stained with a 0.1% ponceau stain following Western anal ysis to control for protein loading. Nitrate and nitrite are the end products of nitric oxide (NO) in vivo Therefore, as an indicator of NOS activity, levels of total nitrate (NO3-) and nitrite (NO2-) were measured in the diaphragm with a colorimetric assay kit (Cayman Chemical, Ann Arbor, MI). Briefly, a section of costal diaphragm was homogenized at a 1: 10 dilution factor in phosphate buffered saline (pH = 7.4). Th e homogenate underwent a series of centrifugations including 10,000 x g for 20 minutes, 100,000 x g for 30 minutes, and 12,000 x g with 10K filter tubes (Millipore Co rporation, Bedford, MA) for 15 minutes. Next, nitrate was converted to nitrite by util izing nitrate reductase. Finally, the addition of the Griess reagents convert s the nitrite into a deep pur ple azo compound. Photometric measurement of the absorbance of this compound de termines the concentration of nitrite.

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21 3-nitrotyrosine Protein levels of 3-nitrotyr osine in the insoluble, cy tosolic, mitochondrial and membrane fractions were measured via Wester n analysis using the protocol described by Barreiro et al. (58). Briefl y, 100 mg of diaphram muscle was homogenized in a buffer containing 10 mM tris-maleate, 3 mM EG TA, 275 mM sucrose, 0.1 mM DTT, and a protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO) (buffer A). One aliquot of homogenate was used for a dot blot while the remaining homogenate was centrifuged at 1000 x g for 10 minutes. The pellet was then resuspended in buffer A and designated as the insoluble fraction. The supernatant was centrifuged at 12,000 x g for 20 min and the pellet was resuspended in buffer B (10 mM tris-maleate, 0.1 mM EDTA, 135 mM KCl). The supernatant was removed and the pellet was resuspended in buffer A and centrifuged at 12,000 x g for 20 min. The pellet was resu spended in buffer A by sonication and was designated as the mitochondrial fraction. Th e supernatant from the last two steps was pooled and centrifuged at 100,000 x g for one hour. The supernatant was saved and designated as the cytosolic fraction while th e pellet was resuspended in buffer C (10 mM HEPES and 300 mM sucrose) and treated for one hour in 600 mM KCl. The homogenate was then centrifuged at 100,000 x g for one hour The pellet was resuspended in buffer A by sonication and designated as the membrane fraction. All four fractions were mixed with sample buffer and boiled for five minut es. Protein was loaded on a 4-20% trisglycine sodium dodecylsulfate (SDS) polyacrylamide gel and separated via electrophoresis (1.5 hours at 100 V) Proteins were then transf erred to nitrocellulose (2 hours at 275 mA) and blocked in 1% BSA. The membrane was exposed to a monoclonal antibody for 3-nitrotyrosin e (Cayman Chemical, Ann Arbor, MI) followed by exposure to a HRP conjugated anti-mouse secondary an tibody. The presence of proteins was

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22 detected using chemiluminescence. Membranes were incubated in SYPRO ruby protein blot stain following Western analysis to control for prot ein loading. Levels of 3nitrotyrosine were normalized to the associated protein band. Statistical Analysis Comparisons between groups were made by a one-way analysis of variance (ANOVA) and, when appropriate, a Tuke y HSD test was performed post hoc. Significance was established at p < 0.05.

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23 CHAPTER 4 RESULTS Systemic and Biologic Response to MV Heart rate (HR) and systol ic blood pressure (BP) were maintained within a physiologic range during the MV protocol (HR = 300 420 beats per minute; BP = 70 130 mm Hg). Arterial pH, the partial pressures of O2 and CO2, and lactate levels were maintained within a normal range during the MV protocol (table 1). Only the SB animals experienced mild hypercapnia and acidosis due to the anesthetic (table 1). Nonetheless, this arterial pH disturban ce did not compromise any of the diaphragmatic contractile properties (see results ). Sodium (Na+), potassium (K+), calcium (Ca++), and glucose levels were also maintained during the SB a nd MV protocol (table 1). Further, there were no significant differences in body weight between the groups prior to the experimental protocol and the 18-hour experime ntal protocol did not alter body weight in any of the groups. This indicates that our hydration and nutrition re gimen was adequate. Also, note that none of the SB or MV animals tested positive for gram-positive or gramnegative bacteria and there were no visual a bnormalities of the lungs or peritoneal cavity. These results indicate that our aseptic surg ical technique was succe ssful in preventing infection. Contractile Dysfunction To determine if apocynin prevented MV-induced diaphragmatic contractile deficits, we measured diaphragmatic c ontractile function in diaphragm strips in vitro Figure 1 illustrates the dia phragmatic force-frequency relationship for all five

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24 experimental groups. Eighteen hours of MV re sulted in a significant reduction (p < 0.05) in the specific force of the diaphragm compar ed to all other groups at all stimulation frequencies except 15 hertz. However, treatme nt with apocynin attenuated all of the MVinduced diaphragmatic dysfunction. Also note that diaphragmatic contractile function in both the SB and the SBA animals did not differ from controls. NADPH Oxidase and MPO Activity Table 2 contains the values for diaphr agmatic NADPH oxidase activity. MV did not result in an increase in NADPH oxidas e activity and apocynin did not reduce the activity below CON values. Importantly, in the absence of the substrate for skeletal muscle NADPH oxidase (NADH), there was almo st no activity (0.8 RLU/mg protein). Also, the addition of superoxide dismutas e (SOD) lowered the activity of control diaphragm homogenate by greater than 60% while the activit y of a positive control (rat liver homogenate) was 20 fold greater than that of control diaphragm. Therefore, this data indicates that our assay was highly specific for NADPH oxidase superoxide production and was not detecting superoxide fr om other sources. Finally, no significant differences were observed betw een groups for myeloperoxidase (MPO) activity (table 2). Glutathione Glutathione is the major non-protein thiol in cells and is considered to be the most important intracellular antioxida nt. MV resulted in a signif icant reduction in the amount of reduced glutathione (GSH) in the diaphrag m, indicative of oxidative stress. Note, however, that diaphragmatic levels of GS H did not differ between the CON and MVA groups (figure 2). Therefore, it appear s that apocynin attenuated the MV-induced oxidative stress in the diaphragm during MV.

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25 Protein Carbonyls Protein carbonyl levels were measured as a general index of protein oxidation in the diaphragm. Compared to all other expe rimental groups (CON, SB, SBA, and MVA), 18 hours of MV resulted in a significant elevatio n in diaphragmatic protein carbonyl levels (figure 3). Indeed, note that the in vivo administration of apocynin prevented the MV-induced increase in prot ein oxidation in the diaphr agm during MV (figure 3). Nitric Oxide Synthase (NOS) MV did not result in a change in diaphr agmatic protein levels of eNOS or nNOS (table 3). Further, iNOS was not detected in the diaphragm of any of the experimental groups. Representative Western blots are illustrated in figu re 4. Finally, MV did not result in an increase in NOS activity as measur ed by the levels of nitrate and nitrite in the diaphragm (table 4). 3-Nitrotyrosine 3-nitrotyrosine is considered to be the primary end product of nitric oxide (NO) interaction with proteins. No significant differences in diaphragmatic levels of 3nitrotyrosine existed between experimental groups in crude homogenate or any of the cellular fractions of diaphragmatic muscle (t able 5-9). Representa tive Western blots of these results are illustrated in figure 5.

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26 Table 1. Arteriole blood values during SB and MV protocol. pH pCO2 (mmHg) pO2 (mmHg) Na+ (mM) K+ (mM) Ca++ (mM) Glucose (mg/dl) Lactate (mM) SB 7.35 0.01 48.7 2.3 62.6 2.9 144.9 0.50 3.47 0.11 1.08 0.02 95.3 4.48 0.51 0.08 MV 7.46 0.01 31.9 1.4 68.5 1.6 143.6 0.53 3.45 0.14 1.05 0.02 96.9 5.37 0.90 0.25 SBA 7.31 0.01 54.2 2.5 71.9 5.1 142.4 0.57 3.55 0.13 1.08 0.03 89.2 5.22 0.47 0.08 MVA 7.41 0.01 35.0 1.7 69.9 2.7 146.5 1.14 3.15 0.09 1.04 0.01 76.3 4.52 0.44 0.06 Values are expressed as mean SEM of the pre, mid and post blood gas samples. CON = control; SB = spontaneously br eathing; MV = mechanically ventilated; SBA = SB with apocynin; MVA = MV with apocynin. Table 2. NADPH oxidase and myeloperoxidase (MPO) activity in the diaphragm. CON SB MV SBA MVA NADPH Oxidase (RLU/mg protein) 392 29 413 23 438 38 354 27 440 42 MPO (U/gww) 1.17 0.069 1.33 0.097 1.22 0.053 1.21 0.046 1.19 0.063 Values are expressed as mean SEM. No significant differenc es were detected between any experimental groups. C ON = control; SB = spontan eously breathing; MV = mechanically ventilated; SBA = SB with apocynin; MVA = MV with apocynin; RLU = relative light units; U/gww = units per gram wet weight. Table 3. Nitric oxide synthase (NOS) protein levels in the diaphragm. CON SB MV eNOS 100.0 8.6 105.2 7.8 91.7 9.9 nNOS 100.0 11.3 108.2 12.2 104.1 14.7 Values are expressed as a percent of control (mean SEM). No significant differences were detected between any of the e xperimental groups. CON = control; SB = spontaneously breathing; MV = mechanically ventilated; eNOS = endothelial nitric oxide synthase; nNOS = neuronal NOS. Table 4. Nitrate and nitrite le vels in the diaphragm. CON SB MV Nitrate and nitrite ( mol/gww) 105.5 9.6 104.5 5.8 99.4 5.9 Values are expressed as mean SEM. No significant differenc es were detected between any experimental groups. C ON = control; SB = spontan eously breathing; MV = mechanically ventilated.

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27 Table 5. Diaphragmatic protein levels of 3-nitrotyrosine in crude homogenate. CON SB MV 100.0 5.9 97.3 5.2 98.6 4.0 Values are expressed as a percent of control (mean SEM). No significant differences existed between any of the experimental groups. CON = control; SB = spontaneously breathing; MV = mechan ically ventilated. Table 6. Diaphragmatic levels of 3-nitr otyrosine within insoluble proteins. ~ Molecular Wt CON SB MV 200 kDa 100.0 7.9 94.8 9.0 88.4 5.7 95 kDa 100.0 11.9 108.3 11.9 96.9 10.0 80 kDa 100.0 5.8 96.7 11.2 109.3 10.3 40 kDa 100.0 9.4 93.9 13.0 102.4 8.2 30 kDa 100.0 9.0 84.7 8.4 109.1 9.7 Values are expressed as a percent of control (mean SEM). No significant differences existed between any of the experimental groups. CON = control; SB = spontaneously breathing; MV = mechan ically ventilated. Table 7. Diaphragmatic protein levels of cytosolic (soluble) 3-nitrotyrosine. ~ Molecular Wt CON SB MV 40 kDa 100.0 7.4 89.5 9.3 86.8 5.3 27 kDa 100.0 6.8 117.9 5.9 109.3 11.1 Values are expressed as a percent of control (mean SEM). No significant differences existed between any of the experimental groups. CON = control; SB = spontaneously breathing; MV = mechan ically ventilated. Table 8. Diaphragmatic mitochondrial prot ein levels of 3-nitrotyrosine. ~ Molecular Wt CON SB MV 70 kDa 100.0 14.6 104.8 13.5 90.5 6.2 40 kDa 100.0 11.9 100.2 13.8 106.0 16.6 22 kDa 100.0 12.4 107.2 7.2 116.1 14.5 Values are expressed as a percent of control (mean SEM). No significant differences existed between any of the experimental groups. CON = control; SB = spontaneously breathing; MV = mechan ically ventilated. Table 9. Diaphragmatic protein levels of 3nitrotyrosine within membrane proteins. ~ Molecular Wt CON SB MV 70 kDa 100.0 10.8 87.8 11.2 102.7 12.8 40 kDa 100.0 14.1 106.1 17.3 100.0 7.6 22 kDa 100.0 13.6 98.7 12.0 108.8 8.5 Values are expressed as a percent of control (mean SEM). No significant differences existed between any of the experimental groups. CON = control; SB = spontaneously breathing; MV = mechan ically ventilated.

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28 Stimulation Frequency (Hz) 020406080100120140160Specific Force (N/cm2) 10 12 14 16 18 20 22 24 26 CON SB SBA MVA MV * * Figure 1. Effect of 18 hours of mechanical ve ntilation (MV) on the diaphragmatic forcefrequency response. Values are expressed as mean SEM. MV significantly different versus all other groups (p< 0.05). CON = control; SB = spontaneously breathing; MV = mechan ically ventilated ; SBA = SB with apocynin; MVA = MV with apocynin.

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29 CONSBMVSBAMVAGlutathione (mmol/gww) 0.0 0.2 0.4 0.6 0.8 # Figure 2. Effect of 18 hours of mechanical ve ntilation on the diaphragmatic levels of reduced glutathione. Values are expressed as mean SEM. Significantly different versus CON (p< 0.05); # Signi ficantly different versus SBA (p < 0.05). Note that the MVA group did not differ from control. CON = control; SB = spontaneously breathing; MV = mechanically ventilated; SBA = SB with apocynin; MVA = MV with apocynin.

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30 CONSBMVSBAMVAProtein Carbonyls (nmol/mg protein) 0.0 0.2 0.4 0.6 0.8 1.0 Figure 3. Effect of 18 hours of mechanical ve ntilation on the diaphragmatic levels of protein carbonyls. Values are expressed as mean SEM. MV significantly different versus all other groups (p < 0.05). Note that the MVA group is significantly lower than the MV group. CON = control; SB = spontaneously breathing; MV = mechanically ventilat ed; SBA = SB with apocynin; MVA = MV with apocynin.

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31 Figure 4. Representative western blots illustra ting the diaphragmatic protein levels of NOS. A) nNOS, B) eNOS, C) iNOS. CON = control; SB = spontaneously breathing; MV = mechanically ventilat ed; +ve = positive control for iNOS (mouse macrophage exposed to IFNand LPS). A) kDa CON SB MV CON SB MV +ve CON SB MV B) kDa C) kDa iNOS nNOS eNOS 130140155

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32 Figure 5. Representative western blots illustra ting the diaphragmatic protein level of 3nitrotyrosine. A) insoluble fraction, B) cytosolic fraction, C) mitochondrial fraction, D) membrane fraction. Arro ws indicate positive band for 3nitrotyrosine. CON = control; SB = spontaneously breathing; MV = mechanically ventilated. A) kDa 250150100755037B) kDa 503725C) kDa 75503725D) kDa 75503725CON SB MV CON SB MV CON SB MV CON SB MV

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33 CHAPTER 5 DISCUSSION Overview of Principal Findings Several important findings emerged from th ese experiments. First, these results indicate that MV-induced oxidative injury to the diaphragm is not due to an increase in NADPH oxidase activity. Secondly, th ese experiments reveal that the in vivo administration of apocynin attenuates the co ntractile dysfunction and oxidative stress induced by MV; however, this amelioration was not due to the inhibition of NADPH oxidase and appears to be due to the antioxi dant properties of apoc ynin. Finally, MV is not associated with an increase in protein le vels of any of the three NOS isoforms (eNOS, nNOS, or iNOS), an increase in NOS activity or an increase in the accumulation of 3nitrotyrosine within the diaphragm. A br ief discussion of these results follows. Role of NADPH Oxidase in MV -induced Oxidative Stress Our first hypothesis for the current expe riment was that NADPH oxidase activity is increased in the diaphragm during MV a nd contributes to diaphragmatic oxidative injury and contractile dysfunction. This postu late was based on data from our laboratory indicating that prolonged MV pr omotes an increase in total calcium levels (unpublished observations) and activation of calcium-activ ated neutral protease s (calpain) in the diaphragm (10). Since NADPH oxidase is a calcium-a ctivated enzyme, we postulated that increases in diaphragma tic free calcium could up-regulate NADPH oxidase activity. Nonetheless, our data do not support the postulate that MV is associated with an increase in NADPH oxidase activity in the diaphragm following MV.

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34 It is important to note that NADPH oxida se exists in two isoforms and that reactive oxygen species (ROS) can be pr oduced by both phagocytic and non-phagocytic isoforms of NADPH oxidase (63). A previ ous investigation from our laboratory has shown that MV is not associated with an increase in phagocytic cells (35). These previous findings agree with the current investigation in th at MV did not result in an increase in myeloperoxidase (MPO) activity in the diaphragm; MPO is considered to be an excellent marker of neutrophil infiltrati on (62). Therefore, based on these collective findings, we conclude that phagocytic NADPH oxidase ac tivity does not increase and apparently does not contribute to MV-induced diaphragmatic dysfunction. The non-phagocytic NADPH oxidase found in skeletal muscle is similar to the phagocytic isoform, however some differen ces do exist. The phagocytic isoform of NADPH oxidase is composed of three cytosolic subunits (p47phox, p67phox, and p40phox) and a membrane-spanning cytochrome b558 composed of two subunits (p22phox and gp91phox). Upon activation, there is a migration of the cy tosolic subunits to the membrane-spanning cytochrome. In comparison, a recent investigation by Javesghani et al. has characterized the nonphagocytic NADPH oxidase enzyme complex in rat skeletal muscle (22). They determined that only four of the five subunits exist in skeletal muscle (p22phox, gp91phox, p47phox, and p67phox) and they are all constitutively expressed on the membrane. Therefore, non-phagocytic NADP H oxidase activation does not require the translocation of subunits from the cytosol to the membrane. Interestingly, the mechanism by which apocynin inhibits phagocytic NADPH oxida se activity is most likely due to the inhibition of the enzyme assembly by bloc king sulfhydryl groups (23). This could

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35 explain, at least in part, why th ere was no inhibitory effect of apocynin on skeletal muscle NADPH oxidase activity in the cu rrent experiments (table 2). Our decision to use apocynin as an NADPH oxidase inhibitor was based on previous investigations show ing its inhibitory effect on NADPH oxidase in skeletal muscle (22, 24). However, dur ing the course of the curr ent experiment, a new report appeared in the literature suggesting th at apocynin has limited efficacy as a nonphagocytic NADPH oxidase in hibitor (64). This report revealed that the in vitro addition of apocynin inhibited ROS formation in phagoc ytic cells (i.e., macrophages) but was not effective in the inhibition of NADPH oxidase activity in non-phagocytic cells (i.e., vascular fibroblasts). If apocynin does not inhibit NADPH oxidase activity in skeletal muscle, why does the administration of apocynin prevent MV-induced diaphragmatic contractile dysfunction? A definitive answer to this question is unavailable but it is possible that apocynin acts as an intracellu lar antioxidant to retard MV -induced oxidative stress and protect against oxidant-mediated diaphragma tic contractile dysf unction. Indeed, the molecular structure of apocynin contains a phenol group with oxidant scavenging capacity. The likelihood that apocynin is a physiological antioxidant could explain our finding that treatment with apocynin preven ted both MV-induced protein oxidation and diaphragmatic contractile dysfunction. The possi bility that apocynin is a physiologically useful antioxidant warrants further inves tigation to determine if this compound is a clinically useful countermeas ure to prevent MV-induced di aphragmatic oxidative stress and contractile dysfunction.

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36 Role of Nitric Oxide Synthase (NOS) in MV-induced Oxidative Stress The second hypothesis tested in this i nvestigation was that NOS levels are elevated during MV, increasing the nitration of diaphragmatic proteins. This postulate was formulated from our preliminary data indi cating that MV results in an increase in total calcium in the diaphragm and knowledge that both of th e constitutive isoforms of NOS (eNOS and nNOS) are calcium-activated enzymes that produce NO. Therefore, an MV-induced increase in calcium could activ ate one or both of these NOS isoforms leading to the formation of NO and the nitrat ion (e.g., 3-nitrotyrosine) of diaphragmatic proteins. Nonetheless, our re sults revealed that 18 hours of MV was not associated with an increase in diaphragmatic le vels NOS or 3-nitrotyrosine. Our finding that MV-induced inactivity in the diaphragm does not alter the levels of any NOS isoform differs from a previous report by Nguyen and Tidball indicating that nNOS levels decrease in mice locomotor skel etal muscle following 10 days of hindlimb unloading (41). Changes in muscle levels of nNOS could be phys iologically significant because an increase in NO in inactive skelet al muscle can be beneficial by inhibiting calpain and, therefore, reducing the degradation of cytoskeletal proteins such as talin and vinculin (65). Note, however, although no ch anges in nNOS levels occurred during 18 hours of MV in the current study, longer periods of MV may decrease levels of nNOS leaving the diaphragm more susceptib le to degradation by calpain. The finding that MV is not associated with an increase in the levels of 3nitrotyrosine in the diaphragm suggests th at NO production was not accelerated in the diaphragm during MV. Our analysis of 3-n itrotyrosine levels in the diaphragm was comprehensive and evaluated the nitration of proteins in a wide variety of protein pools in the cell including the insoluble, cytoso lic, mitochondrial, and membrane protein

PAGE 47

37 fractions of diaphragm fibers following MV. This type of comprehensive analysis is important to detect small treatment-induced ch anges in 3-nitrotyrosin e because a previous investigation by Barreiro et al. has revealed th at the nitration of mu scle proteins can be limited to one or two protein compartments of the muscle fiber (58). For example, these investigators determined that sepsis-induced nitration of proteins was limited to the membrane and mitochondrial fr actions of diaphragm (58). Therefore, the failure to separate muscle proteins into sub-fractions could mask increas es in nitration within small pools of protein within the fiber. However, based upon our comprehensive analysis of 3nitroyrosine in the diaphragm, the current results indicate that 18 hours of MV is not associated with an increase in the nitration of diaphragmatic proteins. Critique of Experimental Model Obtaining a diaphragm muscle biopsy is invasive; therefore, a non-human model must be utilized to study the effects of MV on the diaphragm. The current investigation utilized the rat as the experimental model fo r two reasons: 1) the animal size permits the necessary surgical techniques to be conducted and also allows for the removal of several arterial blood samples which is necessary for the maintenance of blood gas homeostasis, and more importantly, 2) human and rat di aphragms are similar anatomically and functionally as well as having sim ilar fiber type composition (7, 8). Controlled MV was utilized in the cu rrent study versus pressure-assist MV. Complete inactivity of the diaphragm results from controlled MV and is utilized clinically in cases of drug overdose, spinal cord injury, surgery and is also common in pediatric patients (3). Sodium pentobarbital was administered as the anesthetic because the current and previous investigations clearly indicate th at sodium pentobarbital does not compromise

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38 the function of the diaphragm (4, 10, 21). Apocynin was utilized in the current investigation because previous studies have re ported that it is an effective inhibitor of NADPH oxidase in skeletal muscle (22, 24). Another common inhibitor of NADPH oxidase, diphenylene iodonium (DPI), was not u tilized in these experiments because this compound has been shown to be non-selective for NADPH oxidase. Indeed, DPI has been shown to inhibit multiple flavoprote ins (66) including NADPH oxidase (67, 68), NOS (69), xanthine oxidase (70), mito chondrial NADH-ubiquinone oxidoreductase (71, 72), and cytochrome p450 (73). Conclusions These are the first experiments to inves tigate the sources of oxidant production in the diaphragm during prolonged MV. Our studies reveal that MV-induced diaphragmatic dysfunction and oxidative stress is not due to an increase in NADPH oxidase activity. Moreover, the in vivo administration of apocynin attenuat es the diaphragmatic contractile dysfunction and oxidative stress induced by MV via its antioxidant properties. Furthermore, MV does not promote an incr ease in any of the NOS isoforms, NOS activity or cause the nitration of proteins in the diaphragm. Therefore, it can be concluded that neither NADPH oxidase nor NOS cont ributes to diaphragmatic contractile dysfunction and oxidative stress that occurs during MV. Future Directions The current investigation provides evid ence that NADPH oxidase and NOS are not involved in MV-induced diaphragmatic oxi dant stress and contractile dysfunction. However, several other sources of oxidants exis t in skeletal muscle fibers. For example, the mitochondria can produce superoxide at complex I and III along the electron transport chain (44). Although the inves tigation of mitochondrial oxida nts in the diaphragm during

PAGE 49

39 prolonged mechanical ventilation is comp licated, use of mitochondrial targeted antioxidants could be a useful probe to study this phenomenon. Moreover, increases in calcium activat ed neutral proteases (calpain) can upregulate xanthine oxidase activity resulting in the formation of excess superoxide production (44). Our laboratory has reported an increase in calpain activity during MV (10). Therefore, the increas e in calpain activity seen during MV may lead to the production of superoxide via xanthine oxidase. Future experiments using specific pharmacological probes to inhibit xanthine oxidase activity could prove useful in investigating the role of th is oxidant producing pathway. Finally, skeletal muscle atr ophy is associated with an in crease in free, “reactive” iron (48). Free iron can react with hydroge n peroxide and super oxide to produce the highly reactive hydroxyl radical (4 4). Therefore, it is possibl e that MV-induced increases in free iron could be a source of reactive oxygen species in the diaphr agm (10). A variety of membrane permeable iron chelating compounds exist and be useful in investigating the role that reactive iron plays in MV-indu ced oxidant production in the diaphragm. Determining which of the potential sources of oxidant production is involved in MVinduced oxidative stress is important and c ould provide a therapeutic intervention to retard MV-induced diaphragmatic contractile dysfunction.

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40 LIST OF REFERENCES 1. Lemaire F. Difficult weaning. Intensive Care Med 1993;19 Suppl 2:S69-73. 2. Esteban A, Alia I, Ibanez J, Benito S, Tobin MJ. Modes of m echanical ventilation and weaning. A national survey of Spanish hospitals. The Spanish Lung Failure Collaborative Group. Chest 1994;106:1188-93. 3. Hess D, Kacmarek R. 1996. Essentials of mechanical ven tilation. McGraw-Hill, New York. 4. Powers SK, Shanely RA, Coombes JS, Koesterer TJ, McKenzie M, Van Gammeren D, Cicale M, Dodd SL. Mechani cal ventilation resu lts in progressive contractile dysfunction in the diaphragm. J Appl Physiol 2002;92:1851-8. 5. Sassoon CS, Zhu E, Caiozzo VJ. Assist-con trol mechanical ve ntilation attenuates ventilator-induced diaphragmatic dysfunction. Am J Respir Crit Care Med 2004;170:626-32. 6. Metzger JM, Scheidt KB, Fitts RH. Histoc hemical and physiological characteristics of the rat diaphragm. J Appl Physiol 1985;58:1085-91. 7. Mizuno M. Human respiratory muscles: fibre morphology an d capillary supply. Eur Respir J 1991;4:587-601. 8. Powers SK, Demirel HA, Coombes JS, Fletch er L, Calliaud C, Vrabas I, Prezant D. Myosin phenotype and bioenergetic charac teristics of rat respiratory muscles. Med Sci Sports Exerc 1997;29:1573-9. 9. Le Bourdelles G, Viires N, Boczkowski J, Seta N, Pavlovic D, Aubier M. Effects of mechanical ventilation on diaphragmatic contract ile properties in rats. Am J Respir Crit Care Med 1994;149:1539-44. 10. Shanely RA, Zergeroglu MA, Lennon SL, Sugiura T, Yimlamai T, Enns D, Belcastro A, Powers SK. Mechanical vent ilation-induced diaphragmatic atrophy is associated with oxidative injury and increased proteolytic activity. Am J Respir Crit Care Med 2002;166:1369-74. 11. Anzueto A, Peters JI, Tobin MJ, de los Sa ntos R, Seidenfeld JJ, Moore G, Cox WJ, Coalson JJ. Effects of prolonged cont rolled mechanical ventilation on diaphragmatic function in healthy adult baboons. Crit Care Med 1997;25:1187-90.

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41 12. Yang L, Luo J, Bourdon J, Lin MC, Gottfried SB, Petrof BJ. Controlled mechanical ventilation leads to remodeling of the rat diaphragm. Am J Respir Crit Care Med 2002;166:1135-40. 13. Gayan-Ramirez G, de Paepe K, Cadot P, Decramer M. Detrimental effects of shortterm mechanical ventilation on diaphr agm function and IGF-I mRNA in rats. Intensive Care Med 2003;29:825-33. 14. Bernard N, Matecki S, Py G, Lopez S, Mercier J, Capdevila X. Effects of prolonged mechanical ventilation on resp iratory muscle ul trastructure and mitochondrial respir ation in rabbits. Intensive Care Med 2003;29:111-8. 15. Zergeroglu MA, McKenzie MJ, Shanely RA, Van Gammeren D, DeRuisseau KC, Powers SK. Mechanical ventilation-indu ced oxidative stress in the diaphragm. J Appl Physiol 2003;95:1116-24. 16. Dean RT, Fu S, Stocker R, Davies MJ. Biochemistry and pathology of radicalmediated protein oxidation. Biochem J 1997;324 ( Pt 1):1-18. 17. Shringarpure R, Davies KJ. Protein tu rnover by the proteasome in aging and disease. Free Radic Biol Med 2002;32:1084-9. 18. Reznick AZ, Packer L. Oxidative dama ge to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol 1994;233:357-63. 19. Reid MB. Invited Review: redox modulation of skeletal muscle contraction: what we know and what we don't. J Appl Physiol 2001;90:724-31. 20. Powers SK, Demirel HA, Vincent HK, Coombes JS, Naito H, Hamilton KL, Shanely RA, Jessup J. Exercise training im proves myocardial tolerance to in vivo ischemia-reperfusion in the rat. Am J Physiol 1998;275:R1468-77. 21. Betters J, Criswell DS, Shanely RA, Van Gammeren D, Falk DJ, DeRuisseau KC, Deering M, Yimlamai T, Powers SK. Tr olox attenuates mechanical ventilationinduced diaphragmatic dysf unction and proteolysis. Am J Respir Crit Care Med 2004;170:1179-1184. 22. Javesghani D, Magder SA, Barreiro E, Quinn MT, Hussain SN. Molecular characterization of a supe roxide-generating NAD(P)H oxi dase in the ventilatory muscles. Am J Respir Crit Care Med 2002;165:412-8. 23. Simons JM, Hart BA, Ip Vai Ching TR Van Dijk H, Labadie RP. Metabolic activation of natural phenol s into selective oxidative burst agonists by activated human neutrophils. Free Radic Biol Med 1990;8:251-8. 24. Supinski G, Stofan D, Nethery D, Szweda L, DiMarco A. Apocynin improves diaphragmatic function after endotoxin administration. J Appl Physiol 1999;87:776-82.

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42 25. Lawler JM, Song W. Specificity of anti oxidant enzyme inhibition in skeletal muscle to reactive nitr ogen species donors. Biochem Biophys Res Commun 2002;294:1093-100. 26. Criswell DS, Shanely RA, Betters JJ, McKenzie MJ, Sellman JE, Van Gammeren DL, Powers SK. Cumulative effects of aging and mechanical ventil ation on in vitro diaphragm function. Chest 2003;124:2302-8. 27. Radell PJ, Remahl S, Nichols DG, Erik sson LI. Effects of prolonged mechanical ventilation and inactivity on piglet diaphragm function. Intensive Care Med 2002;28:358-64. 28. Sassoon CS, Caiozzo VJ, Manka A, Sieck GC. Altered diaphragm contractile properties with controlled mechanical ventilation. J Appl Physiol 2002;92:2585-95. 29. Watson AC, Hughes PD, Louise Harris M, Hart N, Ware RJ, Wendon J, Green M, Moxham J. Measurement of twitch tr ansdiaphragmatic, esophageal, and endotracheal tube pressure with bilate ral anterolateral magnetic phrenic nerve stimulation in patients in the intensive care unit. Crit Care Med 2001;29:1325-31. 30. Gayan-Ramirez G, Decramer M. Effects of mechanical ventilation on diaphragm function and biology. Eur Respir J 2002;20:1579-86. 31. Racz GZ, Gayan-Ramirez G, Testelmans D, Cadot P, De Paepe K, Zador E, Wuytack F, Decramer M. Early changes in rat diaphragm biology with mechanical ventilation. Am J Respir Crit Care Med 2003;168:297-304. 32. Capdevila X, Lopez S, Bernard N, Rabischong E, Ramonatxo M, Martinazzo G, Prefaut C. Effects of controlled mechan ical ventilation on respiratory muscle contractile properties in rabbits. Intensive Care Med 2003;29:103-10. 33. Shanely RA, Coombes JS, Zergeroglu AM Webb AI, Powers SK. Short-duration mechanical ventilation enhances diaphragma tic fatigue resistance but impairs force production. Chest 2003;123:195-201. 34. Shanely RA, Van Gammeren D, Deruiss eau KC, Zergeroglu AM, McKenzie MJ, Yarasheski KE, Powers SK. Mechanical ve ntilation depresses pr otein synthesis in the rat diaphragm. Am J Respir Crit Care Med 2004;170:994-9. 35. Van Gammeren D, Falk DJ, DeRuisseau KC, Sellman JE, Decramer M, Powers SK. Reloading the diaphragm following m echanical ventilation does not promote injury. Chest 2005;127:2204-2210. 36. Vassilakopoulos T, Petrof BJ. Ventilator-induced diaphragmatic dysfunction. Am J Respir Crit Care Med 2004;169:336-41. 37. Knisely AS, Leal SM, Singer DB. Abnormalities of diaphragmatic muscle in neonates with ventilated lungs. J Pediatr 1988;113:1074-7.

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43 38. Ku Z, Yang J, Menon V, Thomason DB. Decreased polysomal HSP-70 may slow polypeptide elongation during skeletal muscle atrophy. Am J Physiol 1995;268:C1369-74. 39. Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA, Poueymirou WT, Panaro FJ, Na E, Dharmarajan K, Pan ZQ, Valenzuela DM, DeChiara TM, Stitt TN, Yancopoulos GD, Glass DJ. Iden tification of ubiquitin ligases required for skeletal muscle atrophy. Science 2001;294:1704-8. 40. Kandarian SC, Stevenson EJ. Molecular ev ents in skeletal muscle during disuse atrophy. Exerc Sport Sci Rev 2002;30:111-6. 41. Nguyen HX, Tidball JG. Expre ssion of a muscle-specific, nitric oxide synthase transgene prevents muscle membrane injury and reduces muscle inflammation during modified muscle use in mice. J Physiol 2003;550:347-56. 42. Turrens JF. Superoxide production by the mitochondrial re spiratory chain. Biosci Rep 1997;17:3-8. 43. Boveris A, Chance B. The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem J 1973;134:707-16. 44. Halliwell B, Gutteridge J. 1999. Free ra dicals in biology and medicine, 3 ed. Oxford University Press, New York. 45. Kondo H. 2000. Oxidative st ress in muscular atrophy. In C. Sen, L. Packer and O. Hanninen, editors. Handbook of oxidants and antioxidants in exercise. Elsevier Science, St Louis. 46. Kondo H, Kimura M, Itokawa Y. Manganese copper, zinc, and iron concentrations and subcellular distribution in two types of skeletal muscle. Proc Soc Exp Biol Med 1991;196:83-8. 47. Gilbert HF. Biological disulfides: the third messenger? Modulation of phosphofructokinase activity by thiol/disulfide exchange. J Biol Chem 1982;257:12086-91. 48. Kondo H, Miura M, Nakagaki I, Sasaki S, Itokawa Y. Trace element movement and oxidative stress in skeletal muscle atrophied by immobilization. Am J Physiol 1992;262:E583-90. 49. Ryter SW, Tyrrell RM. The heme synthe sis and degradation pathways: role in oxidant sensitivity. Heme oxygenase has both proand antioxidant properties. Free Radic Biol Med 2000;28:289-309. 50. Kondo H, Nakagaki I, Sasaki S, Hori S, Itokawa Y. Mechanism of oxidative stress in skeletal muscle atrophied by immobilization. Am J Physiol 1993;265:E839-44.

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44 51. McCord JM. Oxygen-derived free radi cals in postischemic tissue injury. N Engl J Med 1985;312:159-63. 52. Booth FW, Giannetta CL. Effect of hi ndlimb immobilization upon skeleton muscle calcium in rat. Calcif Tissue Res 1973;13:327-30. 53. DeLeo FR, Quinn MT. Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins. J Leukoc Biol 1996;60:677-91. 54. Griendling KK, Sorescu D, UshioFukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res 2000;86:494-501. 55. Reid MB. Role of nitric oxide in skel etal muscle: synthesi s, distribution and functional importance. Acta Physiol Scand 1998;162:401-9. 56. Stamler JS, Singel DJ, Loscalzo J. Bioc hemistry of nitric oxide and its redoxactivated forms. Science 1992;258:1898-902. 57. Freeman B. Free radical chemistry of nitric oxide. Looking at the dark side. Chest 1994;105:79S-84S. 58. Barreiro E, Comtois AS, Gea J, Laub ach VE, Hussain SN. Protein tyrosine nitration in the ventilatory muscles: role of nitric oxide synthases. Am J Respir Cell Mol Biol 2002;26:438-46. 59. Segal SS, White TP, Faulkner JA. Arch itecture, composition, and contractile properties of rat soleus muscle grafts. Am J Physiol 1986;250:C474-9. 60. Cui XL, Douglas JG. Arachidonic acid act ivates c-jun N-terminal kinase through NADPH oxidase in rabbit proxi mal tubular ep ithelial cells. Proc Natl Acad Sci U S A 1997;94:3771-6. 61. Bradford MM. A rapid and sensitive me thod for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. 62. Seekamp A, Mulligan MS, Till GO, Ward PA. Requirements for neutrophil products and L-arginine in ischemia-reperfusion injury. Am J Pathol 1993;142:1217-26. 63. Bokoch GM, Knaus UG. NADPH oxidases : not just for leukocytes anymore! Trends Biochem Sci 2003;28:502-8. 64. Vejrazka M, Micek R, Stipek S. Apocyni n inhibits NADPH oxidase in phagocytes but stimulates ROS production in non-phagocytic cells. Biochim Biophys Acta 2005;1722:143-7.

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45 65. Koh TJ, Tidball JG. Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells. Am J Physiol Cell Physiol 2000;279:C806-12. 66. Abdelrahman M, Mazzon E, Bauer M, Bauer I, Delbosc S, Cristol JP, Patel NS, Cuzzocrea S, Thiemermann C. Inhibitors of NADPH oxidase reduce the organ injury in hemorrhagic shock. Shock 2005;23:107-14. 67. O'Donnell BV, Tew DG, Jones OT, England PJ. Studies on the inhibitory mechanism of iodonium compounds with sp ecial reference to neutrophil NADPH oxidase. Biochem J 1993;290 ( Pt 1):41-9. 68. Cross AR, Jones OT. The effect of the inhibitor diphenylene iodonium on the superoxide-generating system of neutr ophils. Specific labelling of a component polypeptide of the oxidase. Biochem J 1986;237:111-6. 69. Stuehr DJ, Fasehun OA, Kwon NS, Gross SS, Gonzalez JA, Levi R, Nathan CF. Inhibition of macrophage and endothe lial cell nitric oxi de synthase by diphenyleneiodonium and its analogs. Faseb J 1991;5:98-103. 70. Zhang Z, Blake DR, Stevens CR, Ka nczler JM, Winyard PG, Symons MC, Benboubetra M, Harrison R. A reappraisal of xanthine dehydrogenase and oxidase in hypoxic reperfusion injury: the ro le of NADH as an electron donor. Free Radic Res 1998;28:151-64. 71. Ragan CI, Bloxham DP. Specific labelling of a constituent polypeptide of bovine heart mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone reductase by the inhibitor diphenyleneiodonium. Biochem J 1977;163:605-15. 72. Li Y, Trush MA. Diphenyleneiodonium, an NAD(P)H oxidase inhibitor, also potently inhibits mitochondrial reactive oxygen species production. Biochem Biophys Res Commun 1998;253:295-9. 73. Chen L, Yu LJ, Waxman DJ. Potentiati on of cytochrome P450/cyclophosphamidebased cancer gene therapy by coexpr ession of the P450 reductase gene. Cancer Res 1997;57:4830-7.

PAGE 56

46 BIOGRAPHICAL SKETCH Darin Lee Van Gammeren was born in Luvern e, Minnesota, and raised on a farm in rural Inwood, Iowa. He graduated from the University of Sioux Falls, Sioux Falls, South Dakota, with a bachelor’s de gree in exercise science. In 1999 he moved to Kearney, Nebraska, where he attended the University of Nebraska at Kearney and received a master’s degree in exercise science. Dari n began work on a Doctor of Philosophy degree in exercise physiology in 2001 at the University of Florida. Darin will begin postdoctoral training at Boston University in the ar ea of skeletal muscle disuse atrophy under the direction of Dr. Susan Kandarian in July 2005.


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MECHANISMS OF MECHANICAL VENTRLATION-INDUCED
OXIDATIVE STRESS INT THE DIAPHRAGM













By

DARIN VAN GAMMEREN


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

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Darin Van Gammeren


































This dissertation is dedicated to my parents, Bill and Carol.
















ACKNOWLEDGMENTS

First and foremost I would like to thank my mentor, Dr. Scott Powers, for his

guidance. Without his tutelage, this proj ect would not have been possible and my

education surely lacking. I would also like to thank my committee members, Dr. David

Criswell, Dr. Stephen Dodd, and Dr. Glenn Walter, for their invaluable input. Further, a

big thanks goes to "team diaphragm" (Darin Falk, Melissa Deering, and Dr. Keith

DeRuisseau) for the camaraderie and incalculable assistance on this project. Finally, I

would like to thank Joel French for his technical assistance.





















TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. .................... iv


LI ST OF T ABLE S ................. ................. vii........ ....


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


AB STRAC T ................ .............. ix


CHAPTER


1 INTRODUCTION ................. ...............1.......... ......


2 LITERATURE REVIEW .............. ...............4.....


Mechani cal Ventil ati on-Induced Di aphragmati c Dy sfuncti on ................. .................4
M uscle Force .............. ...............4.....
MV-Induced Atrophy .............. ...............6.....
Structural Injury............... ...............6.
Oxidative Stress ................. ...............8.................
M itochondria ................ ...............9.................
Reactive iron .............. ...............10....
Xanthine oxidase ................ ...............10.................
NADPH oxidase .............. ...............11___ .......

Nitric oxide (NO) ................. ...............11................
Summary ................. ...............13.................


3 M ETHODS ................. ...............15.......... .....


Animals and Experimental Design ................ ...............15................

Experimental Protocol .............. ...............15....
Diaphragmatic Measurements .............. ...............17....
Contractile Measurements ............. ......._......._._ .. ..........1
Biochemical Measurements ...._.._.._ ...... ._._. ...._.._ ............1

NADPH oxidase activity .............. ....__ ....__ ............1
Myeloperoxidase (MPO) activity ................. ................................18
Glutathione ................. ...............19.._._._.......
Protein carbonyl s................. ..............1
Nitric oxide synthase (NOS) .............. ...............19....












3 -nitrotyrosine .........._...._ ...............2_ 1........_....
Statistical Analysis............... ...............22


4 RE SULT S .............. ...............23....


Systemic and Biologic Response to MV ....._.__._ ..... ..._. ...._._. ..........2
Contractile Dysfunction.................... ............2
NADPH Oxidase and MPO Activity .....__.....___ ..........._ ...........2
Glutathione .............. ...............24....
Protein Carbonyl s ................. ...............25...
Nitric Oxide Synthase (NOS) .............. ...............25....
3 -Nitrotyrosine ................. ...............25.................


5 DI SCUS SSION ................. ...............3.. 3......... ....


Overview of Principal Findings ................ .. ........ ..... ...............33......
Role of NADPH Oxidase in MV-induced Oxidative Stress............... .................3
Role of Nitric Oxide Synthase (NOS) in MV-induced Oxidative Stress ...................36
Critique of Experimental Model ................. ...............37................
Conclusions............... ..............3
Future Directions .............. ...............3 8....


LIST OF REFERENCES ................. ...............40........... ....


BIOGRAPHICAL SKETCH .............. ...............46....

















LIST OF TABLES


Table pg

1 Arteriole blood values during SB and MV protocol. ............. .....................2

2 NADPH oxidase and myeloperoxidase (MPO) activity in the diaphragm. .............26

3 Nitric oxide synthase (NOS) protein levels in the diaphragm. ............. .................26

4 Nitrate and nitrite levels in the diaphragm. ............. ...............26.....

6 Diaphragmatic levels of 3 -nitrotyrosine within insoluble proteins. ................... ......27

7 Diaphragmatic protein levels of cytosolic (soluble) 3-nitrotyrosine. .....................27

8 Diaphragmatic mitochondrial protein levels of 3-nitrotyrosine ............... ...............27

9 Diaphragmatic protein levels of 3-nitrotyrosine within membrane proteins. ..........27


















LIST OF FIGURES


Figure pg

1 Effect of 18 hours of mechanical ventilation (MV) on the diaphragmatic force-
frequency response. .............. ...............28....

2 Effect of 18 hours of mechanical ventilation on the diaphragmatic levels of
reduced glutathione. ............. ...............29.....

3 Effect of 18 hours of mechanical ventilation on the diaphragmatic levels of
protein carbonyl s. ............. ...............3 0....

4 Representative western blots illustrating the diaphragmatic protein levels of
NO S ................ ...............31.................

5 Representative western blots illustrating the diaphragmatic protein level of 3-
nitrotyrosine. ............. ...............32.....
















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

MECHANISMS OF MECHANICAL VENTRLATION-INDUCED
OXIDATIVE STRESS INT THE DIAPHRAGM

By

Darin Van Gammeren

August 2005

Chair: Dr. Scott Powers
Major Department: Applied Physiology and Kinesiology

Mechanical ventilation (MV) is associated with oxidative stress and contractile

dysfunction in the diaphragm. The pathways responsible for the production of oxidants

in the diaphragm during MV remain unknown. To address this issue, these experiments

tested the following hypotheses: 1) NADPH oxidase activity is increased during MV and

contributes to the oxidative stress and contractile dysfunction of the diaphragm; and 2)

diaphragmatic nitric oxide synthase (NOS) levels are elevated in the diaphragm during

MV and contribute to nitration of proteins in the diaphragm. To test these postulates, rats

were mechanically ventilated for 18 hours with a subset of animals receiving the NADPH

oxidase inhibitor, apocynin (4 mg/kg body weight). Diaphragmatic NADPH oxidase and

NOS activities were measured along with protein levels of all three NOS isoforms.

Further, 3-nitrotyrosine levels in the diaphragm were measured as an index of protein

nitration. Compared to control, MV resulted in diaphragmatic oxidative stress and a

significant decrease (-10%) in the maximal specific force of the diaphragm. MV did not









increase diaphragmatic NADPH oxidase activity above control. Nonetheless, the

administration of apocynin attenuated MV-induced contractile dysfunction. Interestingly,

treatment with apocynin did not diminish diaphragmatic NADPH oxidase activity but

protected the diaphragm against MV-induced oxidative stress. Moreover, MV did not

promote an increase in diaphragmatic protein levels of eNOS, nNOS, or iNOS or NOS

activity. Consistent with these findings, MV did not elevate diaphragmatic protein levels

of 3-nitrotyrosine in any region of the diaphragm including the insoluble, cytosolic,

mitochondrial, and membrane protein fractions. Therefore, we conclude that MV-

induced oxidative stress in the diaphragm is not due to increases in NADPH oxidase

activity or increased NO production by NOS. Moreover, our results suggest that apocynin

attenuates the MV-induced diaphragm contractile dysfunction and oxidative stress via its

antioxidant properties, not through the inhibition of NADPH oxidase.















CHAPTER 1
INTTRODUCTION

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

patients incapable of doing so on their own. The removal of patients from the ventilator

is termed weaning. Difficulty in weaning is often defined as a weaning procedure

requiring more than 48 hours to permanently remove patients from the ventilator (1).

Importantly, difficulties in weaning occur in approximately 25% of patients utilizing MV

(1). Clinically, this is significant because weaning accounts for greater than 40% of the

total time on the ventilator (2).

There are two major modes of MV: pressure-assist and controlled. As the name

implies, pressure-assist MV assists the patients' inspiratory efforts, while during

controlled MV, the ventilator delivers all of these breaths, therefore rendering the

inspiratory muscles inactive. Pressure-assist MV is commonly utilized in adult patients

suffering from acute respiratory failure whereas controlled MV is utilized in instances

where patients suffer from a spinal cord injury or during surgery (3). Furthermore,

controlled MV is commonly used in pediatric situations (3). Given that controlled MV

completely inactivates the respiratory muscles, as revealed by the absence of EMG

activity in the diaphragm during MV, it is likely that diaphragmatic atrophy and

dysfunction associated with MV occur rapidly using this mode of MV (3-5).

To study the effects of MV on the diaphragm, an animal model must be utilized

because of the invasive nature of removing diaphragm muscle samples. Thus far, four

animal models have been utilized including the baboon, pig, rat, and rabbit. Of these









models, the rat has been utilized in our laboratory due to similarities to the human

diaphragm in fiber type, biochemical properties, anatomical features, and physiological

function (6-8).

Previously, our laboratory has demonstrated a decrement in the specific force of the

diaphragm following as little as 12 hours ofMV (4). Further, this deficit is exacerbated

to a 60% loss in force following 48 hours of MV (9). During MV, our laboratory (10)

and others (9, 11-14) have noted an increase in diaphragmatic atrophy; however, since

the specific force of the diaphragm is normalized per cross sectional area, the decline in

muscle force production is not due to atrophy alone.

One proposed mechanism by which diaphragmatic dysfunction may occur during

MV is via oxidative stress-induced injury to the diaphragm. Our group has found an

increase in protein oxidation and lipid peroxidation following as little as six hours of MV

(10, 15). These oxidized proteins can be targeted by the proteasome proteolytic system

where they are degraded by the 20S proteasome, thereby accelerating muscle atrophy (16,

17). Further, an increase in oxidative stress can damage proteins involved in excitation-

contraction coupling, reducing muscle force production (18-20). The physiological

significance of MV-induced oxidant stress in the diaphragm has been confirmed by

recent experiments demonstrating that infusion of the antioxidant, Trolox, prevents MV-

induced contractile dysfunction in the diaphragm (21).

Various sources of reactive oxygen species (ROS) exist in skeletal muscle

including the calcium-activated enzyme, NADPH oxidase. This source of ROS is

responsible for the one electron reduction of oxygen into superoxide using NADPH or

NADH as the electron donor (22). The inhibition of NADPH oxidase derived superoxide









can be achieved with the addition of apocynin (4-hydroxy-methoxyacetophenone;

acetovanillone). Apocynin most likely reduces superoxide formation by blocking

sulfhydryl groups and inhibiting NADPH oxidase enzyme assembly (23). Previous

investigators have successfully used apocynin in vitro and in vivo to inhibit NADPH

oxidase activity in skeletal muscle (22, 24). NADPH oxidase has been well characterized

in the mammalian diaphragm and remains as a possible source for oxidant production in

inactive skeletal muscle.

Also, nitric oxide (NO) has been reported to be produced in immobilized locomotor

muscle and therefore could also contribute to oxidative injury in the diaphragm during

MV (25). NO is produced by calcium-activated cellular enzyme, nitric oxide synthase

(NOS). NO can react with superoxide to form the highly reactive peroxynitrite molecule

that can nitrosylate proteins in skeletal muscle.

Given that MV-induced oxidant stress is associated with diaphragmatic

contractile dysfunction, determining which oxidant producing pathways are responsible

for the generation of reactive oxygen species is important. Based on preliminary

experiments, we formed the working hypothesis that MV results in an increase in oxidant

production in the diaphragm via increased NADPH oxidase activity and an increase in

nitric oxide synthase (NOS). More specifically, the current experiments are designed to

test two hypotheses: 1) NADPH oxidase activity is increased in the diaphragm during

MV and contributes to diaphragmatic oxidative injury and contractile dysfunction; and 2)

diaphragmatic NOS levels are elevated in the diaphragm during MV and contributes to

the nitration of proteins in the diaphragm.















CHAPTER 2
LITERATURE REVIEW

Mechanical ventilation (MV) is utilized to maintain adequate alveolar ventilation in

patients incapable of doing so on their own. The process of removing patients from the

ventilator is termed weaning, and problems in weaning occur in approximately 25% of

patients exposed to MV for two or more days (1). One of the proposed mechanisms by

which weaning difficulties occur is due to impairments in diaphragmatic strength and

endurance (4, 9, 11-13, 26-29). Importantly, recent experiments in our laboratory suggest

that MV-induced oxidative stress contributes to both diaphragmatic atrophy and

contractile dysfunction (10, 15). Indeed, it is well established that an increase in

oxidative modification of proteins and lipids in the diaphragm can promote skeletal

muscle atrophy and dysfunction (10, 15). This review will outline our current

understanding of MV-induced diaphragm dysfunction and will also provide a brief

overview of oxidant producing pathways that could be responsible for MV-induced

oxidative damage in the diaphragm.

Mechanical Ventilation-Induced Diaphragmatic Dysfunction

Muscle Force

Utilizing a variety of different animal models, diaphragmatic dysfunction has

been evaluated following MV (4, 9-12, 14, 15, 21, 26-28, 30-35). These experiments

reveal that the in vivo transdiaphragmatic pressure of baboons, piglets, and rabbits is

significantly reduced following MV (11, 27, 28). For example, diaphragmatic force

decrements have been shown to occur as early as one day in rabbits and three days in










piglets; these force decrements are exacerbated with time on the ventilator (27, 28).

Further, there is a 40-50% decline in the pressure-generating capacity of the diaphragm

after 3 days in rabbits, 5 days in piglets, and 11 days in baboons (11, 27, 28). Also, it has

been shown that following a prolonged period of MV, animals cannot sustain

diaphragmatic force under an inspiratory resistive load, indicative of an endurance

decrement of the diaphragm (11). Importantly, these impairments in diaphragmatic

function are not attributable to changes in lung volume and abdominal compliance or the

function of the phrenic nerve and neuromuscular junction (1 1, 27).

In addition to the aforementioned in vivo experiments, in vitro preparations have

also been utilized to determine the force of isolated rat and rabbit diaphragm strips

removed from animals following varying periods of MV. A 30-50% reduction in the

maximal isometric specific force of the diaphragm occurs after one to three days of MV

(4, 9, 12, 13, 28) with as little as 12 hours of MV resulting in an approximately 20%

decrement in force (4, 26). The decrements in force cannot be attributed to atrophy alone

due to the fact that the force of the diaphragm in these studies has been normalized to the

cross-sectional area. Furthermore, the diaphragm strip is set at the optimal length;

therefore, the force decrement cannot be due to altered muscle operating length. Finally,

anesthetic agents and neuromuscular blockers are not the cause of the diaphragmatic

dysfunction following MV. This has been experimentally demonstrated by omitting

neuromuscular blockers from several studies and the anesthetic agent has been controlled

by utilizing a spontaneously breathing group of animals under anesthesia, but not

undergoing MV (3 6).









The endurance of the diaphragm following MV has also been assessed in vitro

(13, 28, 32, 33). Conclusions on the fatigue resistance of these diaphragm strips are

inconsistence with studies reporting an increase (32), decrease (33), or no change in the

endurance of the diaphragm following MV (13, 28). Therefore, it is not clear what the

effects of MV are on the endurance of the diaphragm.

In humans, it is much more difficult to study the effects of MV on diaphragmatic

function. Confounding factors such as various disease states, drugs, and modes of

ventilation make it difficult to determine the cause of respiratory muscle dysfunction.

Further, assessment of diaphragm function in humans is complicated given that the

obtainment of diaphragm samples is invasive. However, histopathologic analysis of

diaphragms from 13 neonates ventilated 12 days or more suggests that diaphragm fiber

atrophy occurs (37). Moreover, 33 patients with various diseases exposed to two or more

days of MV exhibited a 50% decrement in the twitch transdiaphragmatic pressure from

supramaximal magnetic stimulation of the phrenic nerve when compared to normal

subjects (29). Therefore, although data from human studies are limited, the current

results are consistent with animal experiments indicating that prolonged MV results in

diaphragmatic atrophy and contractile dysfunction.

MV-Induced Atrophy

MV-induced diaphragm atrophy has been reported in almost all animal experiments

(9, 11-14). MV-induced diaphragmatic atrophy occurs more rapidly (e.g., 18 hours) than

disuse atrophy of peripheral skeletal muscles (9, 10, 12). Disuse muscle atrophy can

occur due to a decrease in protein synthesis (3 8), an increase in protein degradation (39),

or a combination of both. Our laboratory has shown a decrease in protein synthesis

during as little as six hours of MV (34). Moreover, mRNA for insulin-like growth factor









(IGF-1) and type I and IIx myosin heavy chain are depressed after 18 to 24 hours of MV

(13, 34). Importantly, our group has reported an increase in proteolysis after 18 hours of

MV (10). Based upon the relatively long half-life of many skeletal muscle proteins, it is

feasible that the rapid onset of diaphragmatic atrophy is primarily due to a rapid onset of

proteolysis.

There are three primary pathways involved in proteolysis in skeletal muscles: 1)

lysosomal proteases (cathepsins), 2) calcium activated neutral proteases (calpain), and 3)

the proteasome. In regard to protease activation in the diaphragm during MV, Shanely et

al. (10) have reported a greater than twofold increase in diaphragmatic calpain activity

and approximately a fivefold increase in 20S proteasome activity following 18 hours of

MV. Calpains are responsible for the release of myoHilaments from the sarcomere and

allow them to be degraded by the proteasome (40). The proteasome system consists of

the ATP-dependent 26S proteasome, which requires the ubiquitination of proteins prior to

degradation and the 20S proteasome that degrades proteins oxidized by reactive oxygen

species (ROS) without the need for ATP or ubiquitin (36). An increase in calpain-like

activity and 20S proteasome activity has been reported in the diaphragm following MV

(10).

Structural Injury

During MV, alterations in the structure of the diaphragm such as disrupted

myofibrils (14, 28), an increased number of lipid vacuoles in the sarcoplasm and

abnormally small mitochondria with focal membrane disruptions have been reported in

rabbits (14). Investigation on the external intercostals revealed similar findings (14). In

contrast, inactive hindlimb muscles removed from animals undergoing MV do not exhibit

these modifications (28). Myofibril disruption is physiologically significant because an









increase in abnormal myofibrils has been shown to be significantly correlated with

decrements in diaphragmatic force output (28). A definitive explanation for MV-induced

myoHilament disruption does not currently exist. However, at least two possible

explanations exist. First, the calcium-activated protease calpain could be releasing the

myoHilaments from the sarcomere (10). Secondly, periods of spontaneous breathing

could occur during MV that would reload the diaphragm. Reloading of hindlimb muscles

following prolonged periods of disuse has been associated with an increased

susceptibility to muscle fiber injury (41). Nonetheless, we have demonstrated that two

hours of reloading the diaphragm following 24 hours of MV does not exacerbate MV

induced contractile dysfunction or cause membrane damage (35). Therefore, during

periods of 24 hours of MV or less, spontaneous breathing does not appear to contribute to

the structural damage that occurs in the diaphragm.

Oxidative Stress

It is well established that when cellular oxidant production exceeds the capacity of

intracellular antioxidants to scavenge these oxidants, oxidative damage to cellular

biomolecules occurs. In this regard, our research team has reported an increase in protein

oxidation and lipid peroxidation in the diaphragm following various periods of controlled

MV (10, 15). Interestingly, MV-induced oxidative injury occurs in the diaphragm within

as few as six hours after the onset of MV (15). In this study, oxidized proteins were

separated using SDS-PAGE and it was determined that the contractile proteins, actin and

myosin, are oxidized in the diaphragm during prolonged MV (15).

At present, the pathways responsible for MV-induced oxidative stress in the

diaphragm are unknown. Major oxidant producing pathways in cells include the electron









transport chain in the mitochondria, reactive iron, xanthine oxidase, NADPH oxidase,

and nitric oxide synthase. A brief overview of each of these pathways follows.

Mitochondria

Mitochondria primarily function to produce ATP; however, they have also been

shown to produce superoxide radicals (42). The major site of superoxide production

within the mitochondria is the electron transport chain (42). Early components of the

electron transport chain can leak electrons directly onto oxygen while most of the

electrons are transferred to the next component of the chain. It is this leakage of

electrons that generates superoxide (42).

During resting conditions (state four respiration), there is a high degree of

reduction of the electron carriers and a limited supply of ADP as compared to periods of

increased muscle contractile activity (state three respiration) (43). Therefore, during state

four respiration, there is a greater proportional amount of superoxide production and the

production of superoxide has been estimated to account for more than two percent of the

oxygen consumed (43).

Moreover, with increasing concentrations of oxygen, there is an increase in

electron leakage and, therefore, superoxide production (44). However, at physiological

concentrations of oxygen, it has been estimated that only one to three percent of the

oxygen reduced in the mitochondria forms superoxide (44). The low rate of leakage is

most likely due to low oxygen concentrations within the mitochondria and the facilitation

of electron flow by electron carrier complexes. At present, it is unknown if the

mitochondria are a significant source of ROS in the diaphragm during MV.









Reactive iron

The highly reactive hydroxyl radical can be produced from the reaction of

superoxide with hydrogen peroxide, but without reactive metals present, this reaction is

too slow to be of physiological significance (45). In contrast, in the presence of a metal

catalyst like iron or copper, this reaction, termed the Haber-Weiss reaction, will proceed

much more rapidly (45).

During disuse atrophy, muscle fiber volume decreases rapidly and cell structure

changes greatly which can disturb the balance of metals (46, 47). An increase in iron has

been noted throughout 12 days of muscle disuse atrophy as revealed by an increase in the

microsomal fraction of iron (46, 48). Further, an increase in a 54 kDa iron binding

protein in the sarcoplasmic reticulum has been observed as early as four days after the

initiation of skeletal muscle immobilization (46). Finally, when animals were treated

with the iron chelator, deferoxamine, during skeletal muscle immobilization, there was a

decrease in disuse-induced lipid peroxidation and oxidized glutathione (GSSG) in the

immobilized muscles (46). However, when the deferoxamine was saturated with iron,

markers of oxidative stress did not change.

One of the potential sources of free iron is heme oxygenase (HO). HO degrades

heme into iron, carbon monoxide, and biliverdin (49). Of the two isoforms of HO (e.g.,

HO-1 and HO-2), HO-1 is the inducible isoform. Increases in oxidant stress have been

shown to induce HO-1 and HO can serve as a pro or antioxidant (49). By degrading

heme and releasing iron, HO acts as a pro-oxidant. In contrast, HO can also act as an

antioxidant. For example, HO produces biliverdin that can be converted into bilirubin,

and both compounds are antioxidants. Further, HO produces the iron binding protein

ferritin that acts as an antioxidant by binding free iron and therefore preventing the iron-









mediated catalyzed formation of the hydroxyl radical (49). At present, it is unclear if HO

acts as a pro-oxidant or an antioxidant in the diaphragm and this remains as an important

area for future research.

Xanthine oxidase

Xanthine oxidase (XO) is present in the cytoplasm of skeletal muscle and exists in

two forms: NAD-dependent (type D) and superoxide-producing (type O) (50). Both

types have been shown to increase during 12 days of disuse muscle atrophy with

increases in type O being greater (2.3-fold higher). Further, the substrates of XO,

xanthine and hypoxanthine, are increased during muscle disuse as well as their product

(i.e., urate). The larger increase in type O XO increased the ratio of type O XO to total

XO which is indicative of the conversion of type D to type O XO (50). It is known that

this conversion is catalyzed by calcium activated neutral proteases (calpain) (51). This is

consistent with data indicating an increase in intracellular calcium during disuse atrophy

(48, 52). Nonetheless, it is unclear if XO-induced production of oxidants is operative in

the diaphragm during prolonged MV.

NADPH oxidase

NADPH oxidase is a membrane-associated enzyme that produces superoxide via a

one-electron reduction of oxygen using NADPH or NADH as the electron donor. The

enzyme exists in phagocytes (53) and nonphagocytes. Several differences exist between

NADPH oxidases found in phagocytic and nonphagocytic cells including enzyme

orientation, direction of superoxide production, subunit structure, and substrate

preference (54). The phagocytic NADPH oxidase consists of five subunits including a

plasma membrane-spanning cytochrome b558 that is composed of the p22phox and

gp91phox subunits. Also, there are three subunits located in the cytosol including the









p47phox, p67phox, and p40phox. The cytosolic subunits are not associated with the

membrane bound cytochrome until activation. Upon activation, the utilization of

intracellular NADPH or NADH causes the transfer of electrons to oxygen in the

extracellular space, producing superoxide outside of the cell (22).

Recently, it was discovered by Javesghani et al. (22) that the p22phox, gp91phox,

p67phox, and p47phox subunits' mRNA and protein were located in skeletal muscle

while p40phox was only in the blood vessels. Furthermore, this group discovered that the

four subunits are constitutively expressed in the membrane, unlike the phagocytic

NADPH oxidase. Importantly, they demonstrated that NADPH oxidase was capable of

significant level of superoxide production in skeletal muscle. Given that NADPH

oxidase can be activated by increases in intracellular calcium levels, we postulate that

NADPH oxidase is a possible source of oxidant production in the diaphragm during

prolonged MV.Nitric oxide (NO)

Nitric oxide (NO)

The free radical, nitric oxide (NO), is produced by the enzyme NO synthase

(NOS). There are three isoforms of NOS: 1) type I or neuronal (nNOS), 2) type II or

inducible (iNOS), and 3) type III or endothelial (eNOS). All forms of NOS produce NO

from L-arginine and require oxygen and NADPH as substrates while citrulline is a

byproduct (55). The cofactors involved in this reaction include FAD, FMN,

tetrahydrobiopterin, heme, and calmodulin. The binding of NOS and calmodulin is

calcium dependent with only the constitutive isoforms (nNOS and eNOS) being calcium

sensitive (55). The calcium sensitive binding of calmodulin is the primary regulator of

the production of NO via the constitutive isoforms of NOS; however, iNOS is tightly









bound to calmodulin and not calcium regulated. iNOS is primarily regulated at the

transcriptional level and is upregulated due to an inflammatory challenge (55).

Targets of NO can be generalized into three main categories. First, NO can react

with oxygen and superoxide to form low molecular weight NO derivatives such as

peroxynitrite. These molecules maintain redox activity and can participate in the transfer

of electrons (56). The NO derivative, peroxynitrite, is one of the most reactive free

radicals involved in oxidative damage within skeletal muscle (57). Second, derivatives of

NO can react with transition metals such as iron to produce NO-metal adducts. This is a

mechanism by which NO modulates metalloprotein function. Third, one of the main

targets of NO is reduced thiols. NO can cause the S-nitrosylation of protein thiols, which

is reversible (55). The formation of 3-nitrotyrosine is one of the most commonly studied

covalent modifications of proteins by NO (58).

Summary

Previous work has demonstrated that control MV renders the diaphragm

completely inactive. During this period of disuse, diaphragmatic force decrements occur

within as little as 12 hours of MV. Further, diaphragmatic atrophy occurs but cannot

account for all of the dysfunction due to the fact that the force of the diaphragm is

normalized per cross sectional area.

MV has also been associated with an increase in oxidative stress in the

diaphragm. Specifically, following as few as six hours of MV, increased lipid

peroxidation and protein oxidation occurs within the diaphragm. This is physiologically

significant because MV-induced oxidative stress can accelerate diaphragmatic proteolysis

and contractile dysfunction. Numerous biochemical pathways could be involved in the

increase in oxidants that cause damage to the diaphragm including the mitochondria, free









(reactive) iron, xanthine oxidase, NADPH oxidase, and NO. While all of these pathways

are potential contributors to the increase in oxidative stress that occurs during MV, the

primary aim of this investigation is to determine if the NADPH oxidase pathway and NO

pathway are involved in MV-induced oxidative injury in the diaphragm. Our long-term

obj ective is to determine which ROS pathways are involved in oxidant production in the

diaphragm during prolonged MV and to develop specific antioxidant countermeasures to

protect the diaphragm against the detrimental effects of MV.















CHAPTER 3
METHOD S

Animals and Experimental Design

These experiments were approved by the University of Florida Animal Care and

Use Committee and followed the guidelines for animal experiments set forth by the

National Institutes of Health. Female, Sprague-Dawley rats (4-months old) were

randomly assigned to one of five groups, n = 8 per group: 1) acute control, 2) 18 hour

spontaneously breathing control (SB), 3) 18 hour SB control with NADPH oxidase

inhibition (SBA), 4) 18 hour mechanically ventilated (MV), 5) 18 hour MV with NADPH

oxidase inhibition (MVA).

Experimental Protocol

Animals were anesthetized with sodium pentobarbital (60 mg/kg body weight,

intraperitoneal (IP)). After reaching a surgical plane of anesthesia, the acute control

animals were sacrificed immediately while the SB and MV animals were

tracheostomized utilizing aseptic techniques. The SB animals breathed spontaneously for

the 18-hour duration while the MV animals were mechanically ventilated with a volume-

driven ventilator (Inspira, Harvard Apparatus, Cambridge, MA) for the same duration.

The tidal volume was set at approximately 0.55 ml/100 grams body weight with a

respiratory rate of 80 breaths per minute and a positive end-expiratory pressure (PEEP) of

one centimeter water. In the SBA and MVA animals, the NADPH oxidase inhibitor,

apocynin, was dissolved in saline and administered via an IP inj section prior to the









experimental protocol (4 mg/kg body weight). This dosage has been previously utilized

in vivo to inhibit NADPH oxidase activity in skeletal muscle (24).

The carotid artery was cannulated to permit measurement of arterial blood

pressure and the collection of blood. During the first hour of the experimental protocol,

after approximately 9 hours and during the final hour of the experiment, blood samples

were analyzed for the partial pressures of 02 and CO2, arterial pH, sodium (Na ),

potassium (K ), calcium (Ca++), glucose and lactate using an electronic blood gas

analyzer (GEM Premier 3000; Instrumentation Laboratory, Lexington, MA). Arterial

PO2 WAS maintained throughout the experiment by gradually increasing the FIO2 USing a

hyperoxic gas (range 22-25% oxygen) in both SB and MV animals. Moreover, the

jugular vein was cannulated for the infusion of saline and sodium pentobarbital (~10

mg/kg body weight/hour). Apocynin was administered via an IP inj section (4 mg/kg body

weight) .

Furthermore, animals received an intramuscular inj section of glycopyrrolate (0.04

mg/kg body weight) every two hours to reduce airway secretions. Body temperature was

maintained at approximately 37oC and heart rate was monitored via a lead II

electrocardiograph. Continuous care during the experimental protocol included

lubricating the eyes, expressing the bladder, removing airway mucus and rotating the

animal and limbs of the animal. Enteral nutrition was provided via the AIN-76 rodent

diet with a nutrient composition of 15% proteins, 35% lipids, 50% carbohydrates, and

vitamins and minerals (Research Diets Inc., New Brunswick, NJ). Our planned feeding

schedule was designed to provide an isocaloric diet with the nutrients administered every









two hours with a gastric tube; the total administration of 69 ml is equivalent to 69

kcal/day .

Following the experimental protocol, the diaphragm was quickly removed and

placed in a dissecting chamber containing Krebs-Hensleit saline aerated with 95/5%

02/CO2. One segment of the costal diaphragm was used to assess the in vitro contractile

function of the diaphragm while the remaining costal diaphragm was dissected into

multiple segments (~ 50 mg) and quickly frozen in liquid nitrogen and stored at -80oC

for subsequent assay. Finally, due to the negative effect of sepsis on the diaphragm,

blood samples from each animal were cultured to determine if gram positive and gram

negative bacteria were present in the blood.

Diaphragmatic Measurements

Contractile Measurements

The force-frequency response of a strip of costal diaphragm was performed and

normalized to the cross-sectional area (CSA) as described previously (4). Briefly, a strip

of diaphragm muscle was obtained from the midcostal region including the tendinous

attachments at the central tendon and rib cage. The strip was vertically suspended in a

jacked tissue bath between two plexiglas clamps with one end connected to an isometric

force transducer (model FT-03, Grass Instruments, Quincy, MA). The tissue bath was

filled with Krebs-Hensleit saline (pH = 7.4) aerated with 95/5% 02/CO2.

Following a 15-minute equilibration period at 25oC, the muscle strip was

stimulated with platinum electrodes surrounding the muscle strip. A supramaximal

stimulation voltage (~150%) was utilized to determine optimal contractile length (Lo) of

the muscle strip by systematically adjusting the length of the muscle while stimulating it

with single twitches. All contractile measurements were made at Lo.









To determine the force-frequency response, each muscle strip was stimulated with

120 V pulses at 15-160 Hz with a train duration of 500 ms. Contractions were separated

by a two minute recovery period. Following this protocol, the muscle strip length was

measured at Lo using a caliper. The strip was then trimmed from the supporting rib and

all connective tissue and fat was removed. The remaining strip of muscle was weighed

and the CSA was determined by using the following formula: total muscle CSA (mm2)

[muscle mass/(fiber length x 1. 056)]. The density of muscle in g/cm3 is 1.056 (59).

Biochemical Measurements

NADPH oxidase activity

The activity of NADPH oxidase was measured using a lucigenin technique as

described by Cui and Douglas (60). Briefly, muscle was homogenized in a 20 mM

potassium phosphate buffer (KPO4) (pH = 7.2) containing 1 mM EGTA. One hundred

microliters of supernatant was added to 900 ul of buffer (50 mM KPO4, 1 mM EGTA,

150 mM sucrose, 230 uM lucigenin, and 500 uM NADH). The change in luminescence

was measured over a 10-minute period and normalized to protein. Protein concentration

of the homogenate was determined using the Bradford technique (61).

Myeloperoxidase (MPO) activity

MPO has been shown to be highly correlated with the number of neutrophils

present in the tissue and therefore MPO activity was determined as an indication of the

level of neutrophil infiltration into the diaphragm during our experiments (62). MPO

activity was measured using the method of Seekamp et al. (62). Briefly, muscle was

homogenized in a 50 mM KPO4 buffer containing 0.5% HTAB and 5 mM EDTA (pH =

6.0). Homogenate was sonicated for 10 seconds and centrifuged cold at 3000 x g for 30

minutes. Ten microliters of homogenate was added to 290 ul of the reaction solution (50









mM KPO4 buffer, 3% H202, 1% O-dianisidine, pH = 6.0). The change in absorbance was

measured for three minutes and the value was expressed as activity units per gram of

muscle (wet weight).

Glutathione

As a marker of oxidative stress, reduced glutathione levels were measured in a

segment of costal diaphragm using a commercially available kit (Cayman Chemical, Ann

Arbor, MI). The principal of this assay is that the sulfhydryl group of glutathione (GSH)

reacts with 5,5-dithiobis-2-nitrobenzoic acid (DTNB) to produce a yellow-colored 5-thio-

2-nitrobenzoic acid (TNB). The mixed disulfide that is produced by this reaction is

reduced by glutathione reductase to recycle the GSH and produce more TNB. The rate of

TNB production is directly proportional to the recycling reaction and, therefore,

reflective of the concentration of GSH in the sample. Reduced GSH was calculated by

subtracting the amount of oxidized glutathione (GS SG) from the amount of total

glutathione. The reduced form of GSH is the most prevalent in biological systems.

Protein carbonyls

The carbonyl assay is a general assay of oxidative damage to proteins. The

principle of the assay is that several reactive oxygen species attack amino acid residues in

proteins to produce products with carbonyl groups which can be measured after reaction

with 2,4-dinitrophenylhydrazine. To measure protein carbonyl levels in our muscle

samples, a segment of costal diaphragm was homogenized in phosphate buffered saline

(pH = 7.4) and centrifuged at 1000 x g for 30 minutes. The supernatant was adjusted to

contain two mg protein/ml and reacted with 2,4-dinitrophenylhydrazine overnight.

Protein carbonyl levels in each sample were then detected via a commercially available

ELISA kit (Zenith Technology Corporation, Dunedin, NZ).









Nitric oxide synthase (NOS)

Protein levels of all three isoforms of NOS (eNOS, nNOS, iNOS) were detected by

Western analysis. Crude muscle homogenate (75 ug protein) was loaded onto a 7.5%

tris-glycine SDS polyacrylamide gel and separated via electrophoresis (100 V, 1.5 hours).

Proteins were then transferred to nitrocellulose (2 hours at 275 mA) and the membrane

was blocked in 5% non-fat dry milk. The membrane was then exposed to a monoclonal

antibody for eNOS, nNOS, and iNOS at a dilution of 1:500 (BD Transduction

Laboratories, Lexington, KY) followed by exposure to a HRP conjugated anti-mouse

secondary antibody. Positive controls were included on each gel including human

endothelial cell lysate, rat cerebrum lysate, and mouse macrophage stimulated with

IFNy/LPS for eNOS, nNOS, and iNOS, respectively. Further, membranes were stained

with a 0.1% ponceau stain following Western analysis to control for protein loading.

Nitrate and nitrite are the end products of nitric oxide (NO) in vivo. Therefore, as

an indicator of NOS activity, levels of total nitrate (NO3-) and nitrite (NO2-) were

measured in the diaphragm with a colorimetric assay kit (Cayman Chemical, Ann Arbor,

MI). Briefly, a section of costal diaphragm was homogenized at a 1:10 dilution factor in

phosphate buffered saline (pH = 7.4). The homogenate underwent a series of

centrifugations including 10,000 x g for 20 minutes, 100,000 x g for 30 minutes, and

12,000 x g with 10K filter tubes (Millipore Corporation, Bedford, MA) for 15 minutes.

Next, nitrate was converted to nitrite by utilizing nitrate reductase. Finally, the addition

of the Griess reagents converts the nitrite into a deep purple azo compound. Photometric

measurement of the absorbance of this compound determines the concentration of nitrite.









3-nitrotyrosine

Protein levels of 3-nitrotyrosine in the insoluble, cytosolic, mitochondrial and

membrane fractions were measured via Western analysis using the protocol described by

Barreiro et al. (58). Briefly, 100 mg of diaphram muscle was homogenized in a buffer

containing 10 mM tris-maleate, 3 mM EGTA, 275 mM sucrose, 0.1 mM DTT, and a

protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) (buffer A). One aliquot of

homogenate was used for a dot blot while the remaining homogenate was centrifuged at

1000 x g for 10 minutes. The pellet was then resuspended in buffer A and designated as

the insoluble fraction. The supernatant was centrifuged at 12,000 x g for 20 min and the

pellet was resuspended in buffer B (10 mM tris-maleate, 0.1 mM EDTA, 135 mM KC1).

The supernatant was removed and the pellet was resuspended in buffer A and centrifuged

at 12,000 x g for 20 min. The pellet was resuspended in buffer A by sonication and was

designated as the mitochondrial fraction. The supernatant from the last two steps was

pooled and centrifuged at 100,000 x g for one hour. The supernatant was saved and

designated as the cytosolic fraction while the pellet was resuspended in buffer C (10 mM

HEPES and 300 mM sucrose) and treated for one hour in 600 mM KC1. The homogenate

was then centrifuged at 100,000 x g for one hour. The pellet was resuspended in buffer A

by sonication and designated as the membrane fraction. All four fractions were mixed

with sample buffer and boiled for five minutes. Protein was loaded on a 4-20% tris-

glycine sodium dodecylsulfate (SDS) polyacrylamide gel and separated via

electrophoresis (1.5 hours at 100 V). Proteins were then transferred to nitrocellulose (2

hours at 275 mA) and blocked in 1% BSA. The membrane was exposed to a monoclonal

antibody for 3-nitrotyrosine (Cayman Chemical, Ann Arbor, MI) followed by exposure to

a HRP conjugated anti-mouse secondary antibody. The presence of proteins was









detected using chemiluminescence. Membranes were incubated in SYPRO ruby protein

blot stain following Western analysis to control for protein loading. Levels of 3-

nitrotyrosine were normalized to the associated protein band.

Statistical Analysis

Comparisons between groups were made by a one-way analysis of variance

(ANOVA) and, when appropriate, a Tukey HSD test was performed post hoc.

Significance was established at p < 0.05.















CHAPTER 4
RESULTS

Systemic and Biologic Response to MV

Heart rate (HR) and systolic blood pressure (BP) were maintained within a

physiologic range during the MV protocol (HR = 300 420 beats per minute; BP = 70 -

130 mm Hg). Arterial pH, the partial pressures of 02 and CO2, and lactate levels were

maintained within a normal range during the MV protocol (table 1). Only the SB animals

experienced mild hypercapnia and acidosis due to the anesthetic (table 1). Nonetheless,

this arterial pH disturbance did not compromise any of the diaphragmatic contractile

properties (see results). Sodium (Na ), potassium (K ), calcium (Ca +), and glucose

levels were also maintained during the SB and MV protocol (table 1). Further, there

were no significant differences in body weight between the groups prior to the

experimental protocol and the 18-hour experimental protocol did not alter body weight in

any of the groups. This indicates that our hydration and nutrition regimen was adequate.

Also, note that none of the SB or MV animals tested positive for gram-positive or gram-

negative bacteria and there were no visual abnormalities of the lungs or peritoneal cavity.

These results indicate that our aseptic surgical technique was successful in preventing

infection.

Contractile Dysfunction

To determine if apocynin prevented MV-induced diaphragmatic contractile

deficits, we measured diaphragmatic contractile function in diaphragm strips in vitro.

Figure 1 illustrates the diaphragmatic force-frequency relationship for all five









experimental groups. Eighteen hours ofMV resulted in a significant reduction (p < 0.05)

in the specific force of the diaphragm compared to all other groups at all stimulation

frequencies except 15 hertz. However, treatment with apocynin attenuated all of the MV-

induced diaphragmatic dysfunction. Also note that diaphragmatic contractile function in

both the SB and the SBA animals did not differ from controls.

NADPH Oxidase and MPO Activity

Table 2 contains the values for diaphragmatic NADPH oxidase activity. MV did

not result in an increase in NADPH oxidase activity and apocynin did not reduce the

activity below CON values. Importantly, in the absence of the substrate for skeletal

muscle NADPH oxidase (NADH), there was almost no activity (0.8 RLU/mg protein).

Also, the addition of superoxide dismutase (SOD) lowered the activity of control

diaphragm homogenate by greater than 60% while the activity of a positive control (rat

liver homogenate) was 20 fold greater than that of control diaphragm. Therefore, this

data indicates that our assay was highly specific for NADPH oxidase superoxide

production and was not detecting superoxide from other sources. Finally, no significant

differences were observed between groups for myeloperoxidase (MPO) activity (table 2).

Glutathione

Glutathione is the maj or non-protein thiol in cells and is considered to be the most

important intracellular antioxidant. MV resulted in a significant reduction in the amount

of reduced glutathione (GSH) in the diaphragm, indicative of oxidative stress. Note,

however, that diaphragmatic levels of GSH did not differ between the CON and MVA

groups (figure 2). Therefore, it appears that apocynin attenuated the MV-induced

oxidative stress in the diaphragm during MV.









Protein Carbonyls

Protein carbonyl levels were measured as a general index of protein oxidation in

the diaphragm. Compared to all other experimental groups (CON, SB, SBA, and MVA),

18 hours of MV resulted in a significant elevation in diaphragmatic protein carbonyl

levels (figure 3). Indeed, note that the in vivo administration of apocynin prevented the

MV-induced increase in protein oxidation in the diaphragm during MV (Higure 3).

Nitric Oxide Synthase (NOS)

MV did not result in a change in diaphragmatic protein levels of eNOS or nNOS

(table 3). Further, iNOS was not detected in the diaphragm of any of the experimental

groups. Representative Western blots are illustrated in Eigure 4. Finally, MV did not

result in an increase in NOS activity as measured by the levels of nitrate and nitrite in the

diaphragm (table 4).

3-Nitrotyrosine

3-nitrotyrosine is considered to be the primary end product of nitric oxide (NO)

interaction with proteins. No significant differences in diaphragmatic levels of 3-

nitrotyrosine existed between experimental groups in crude homogenate or any of the

cellular fractions of diaphragmatic muscle (table 5-9). Representative Western blots of

these results are illustrated in figure 5.









Table 1. Arteriole blood values during SB and MV protocol.
pH pCO2 pO2 Na+ K+ Ca+ Glucose Lactate
(mmHg) (mmHg) (mM) (mM) (mM) (mg/dl) (mM)
SB 7.35 f 48.7 f 62.6 f 144.9 f 3.47 f 1.08 f 95.3 f 0.51 f
0.01 2.3 2.9 0.50 0.11 0.02 4.48 0.08
MV 7.46 f 31.9 f 68.5 f 143.6 f 3.45 f 1.05 f 96.9 f 0.90 f
0.01 1.4 1.6 0.53 0.14 0.02 5.37 0.25
SBA 7.31 f 54.2 f 71.9 f 142.4 f 3.55 f 1.08 f 89.2 f 0.47 f
0.01 2.5 5.1 0.57 0.13 0.03 5.22 0.08
MVA 7.41 f 35.0 f 69.9 f 146.5 f 3.15 f 1.04 f 76.3 f 0.44 f
0.01 1.7 2.7 1.14 0.09 0.01 4.52 0.06
Values are expressed as mean f SEM of the pre, mid and post blood gas samples. CON =
control; SB = spontaneously breathing; MV = mechanically ventilated; SBA = SB with
apocynin; MVA = MV with apocynin.

Table 2. NADPH oxidase and myeloperoxidase (MPO) activity in the diaphragm.
CON SB MV SBA MVA
NADPH Oxidase 392 f 413 f 438 f 354 f 440 f
(RLU/mg protein) 29 23 38 27 42
MPO (U/gww) 1.17 & 1.33 f 1.22 f 1.21 f 1.19 &
0.069 0.097 0.053 0.046 0.063
Values are expressed as mean f SEM. No significant differences were detected between
any experimental groups. CON = control; SB = spontaneously breathing; MV =
mechanically ventilated; SBA = SB with apocynin; MVA = MV with apocynin; RLU =
relative light units; U/gww = units per gram wet weight.

Table 3. Nitric oxide synthase (NOS) protein levels in the diaphragm.
CON SB MV
eNOS 100.0 f 8.6 105.2 f 7.8 91.7 f 9.9
nNOS 100.0 f 11.3 108.2 f 12.2 104.1 f 14.7

Values are expressed as a percent of control (mean f SEM). No significant differences
were detected between any of the experimental groups. CON = control; SB =
spontaneously breathing; MV = mechanically ventilated; eNOS = endothelial nitric oxide
synthase; nNOS = neuronal NOS.

Table 4. Nitrate and nitrite levels in the diaphragm.
CON SB MV
Nitrate and nitrite (Clmol/gww) 105.5 f 9.6 104.5 f 5.8 99.4 f 5.9
Values are expressed as mean f SEM. No significant differences were detected between
any experimental groups. CON = control; SB = spontaneously breathing; MV =
mechanically ventilated.




















































Table 9. Diaphragmatic protein levels of 3-nitrotyrosine within membrane proteins.
~ Molecular Wt CON SB MV
70 kDa 100.0 f 10.8 87.8 f 11.2 102.7 f 12.8
40 kDa 100.0 f 14.1 106.1 f 17.3 100.0 f 7.6
22 kDa 100.0 f 13.6 98.7 f 12.0 108.8 f 8.5
Values are expressed as a percent of control (mean f SEM). No significant differences
existed between any of the experimental groups. CON = control; SB = spontaneously
breathing; MV = mechanically ventilated.


Table 5. Diaphragmatic protein levels of 3-nitrotyrosine in crude homogenate.
CON SB MV
100.0 f 5.9 97.3 f 5.2 98.6 f 4.0
Values are expressed as a percent of control (mean f SEM). No significant differences
existed between any of the experimental groups. CON = control; SB = spontaneously
breathing; MV = mechanically ventilated.

Table 6. Diaphragmatic levels of 3 -nitrotyrosine within insoluble proteins.
~ Molecular Wt CON SB MV
200 kDa 100.0 f 7.9 94.8 f 9.0 88.4 f 5.7
95 kDa 100.0 f 11.9 108.3 f 11.9 96.9 f 10.0
80 kDa 100.0 f 5.8 96.7 f 11.2 109.3 f 10.3
40 kDa 100.0 f 9.4 93.9 f 13.0 102.4 f 8.2
30 kDa 100.0 f 9.0 84.7 f 8.4 109.1 f 9.7
Values are expressed as a percent of control (mean f SEM). No significant differences
existed between any of the experimental groups. CON = control; SB = spontaneously
breathing; MV = mechanically ventilated.

Table 7. Diaphragmatic protein levels of cytosolic (soluble) 3-nitrotyrosine.
~ Molecular Wt CON SB MV
40 kDa 100.0 f 7.4 89.5 f 9.3 86.8 f 5.3
27 kDa 100.0 f 6.8 117.9 f 5.9 109.3 f 11.1
Values are expressed as a percent of control (mean f SEM). No significant differences
existed between any of the experimental groups. CON = control; SB = spontaneously
breathing; MV = mechanically ventilated.


Table 8. Diaphragmatic mitochondrial protein levels of 3-nitrotyrosine.
~ Molecular Wt CON SB MV


70 kL~a 100.0 f 14.6 104.8 f 13.5 90.5 f 6.2
40 kDa 100.0 f 11.9 100.2 f 13.8 106.0 f 16.6
22 kDa 100.0 f 12.4 107.2 f 7.2 116.1 f 14.5
Values are expressed as a percent of control (mean f SEM). No significant differences
existed between any of the experimental groups. CON = control; SB = spontaneously
breathing; MV = mechanically ventilated.










26-
E 24
z 22 f
Q~20 -*CO
o 18 -* sB
.9 16 MA


co 12 M



0 20 40 60 80 1 00 1 20 1 40 1 60

Stimulation Freq uency (Hz)

Figure 1. Effect of 18 hours of mechanical ventilation (MV) on the diaphragmatic force-
frequency response. Values are expressed as mean + SEM. MV significantly
different versus all other groups (p< 0.05). CON = control; SB =
spontaneously breathing; MV = mechanically ventilated; SBA = SB with
apocynin; MVA = MV with apocynin.










0.8



0.6











0.0
CON SB MV SBA MVA


Figure 2. Effect of 18 hours of mechanical ventilation on the diaphragmatic levels of
reduced glutathione. Values are expressed as mean + SEM. Significantly
different versus CON (p< 0.05); # Significantly different versus SBA (p <
0.05). Note that the MVA group did not differ from control. CON = control;
SB = spontaneously breathing; MV = mechanically ventilated; SBA = SB
with apocynin; MVA = MV with apocynin.










1.0








.50.4

o E
& 5 0.2


0.0
CON SB MV SBA MVA


Figure 3. Effect of 18 hours of mechanical ventilation on the diaphragmatic levels of
protein carbonyls. Values are expressed as mean + SEM. MV significantly
different versus all other groups (p< 0.05). Note that the MVA group is
significantly lower than the MV group. CON = control; SB = spontaneously
breathing; MV = mechanically ventilated; SBA = SB with apocynin; MVA =
MV with apocynin.










I I


A) kDa CON SB
155- ..; a .


MV


MV.....
A eNOS


MV +ve

-I iNOS


Figure 4. Representative western blots illustrating the diaphragmatic protein levels of
NOS. A) nNOS, B) eNOS, C) iNOS. CON = control; SB = spontaneously
breathing; MV = mechanically ventilated; +ve = positive control for iNOS
(mouse macrophage exposed to IFN-yI and LPS).


InNOS


B) kDa CON SB
140-1

C) kDa CON SB
130-





A) kDa
250-


150-

100-
75-

50-
37-


B) kDa
50- CON


CON SB MV


SB


MV


C) kDa CN
75-


SB MV


D) kDa
75-

50
37-

25


50-
37-

25-


Figure 5. Representative western blots illustrating the diaphragmatic protein level of 3-
nitrotyrosine. A) insoluble fraction, B) cytosolic fraction, C) mitochondrial
fraction, D) membrane fraction. Arrows indicate positive band for 3-
nitrotyrosine. CON = control; SB = spontaneously breathing; MV =
mechanically ventilated.


CON SB MV















CHAPTER 5
DISCUSSION

Overview of Principal Findings

Several important findings emerged from these experiments. First, these results

indicate that MV-induced oxidative injury to the diaphragm is not due to an increase in

NADPH oxidase activity. Secondly, these experiments reveal that the in vivo

administration of apocynin attenuates the contractile dysfunction and oxidative stress

induced by MV; however, this amelioration was not due to the inhibition of NADPH

oxidase and appears to be due to the antioxidant properties of apocynin. Finally, MV is

not associated with an increase in protein levels of any of the three NOS isoforms (eNOS,

nNOS, or iNOS), an increase in NOS activity or an increase in the accumulation of 3-

nitrotyrosine within the diaphragm. A brief discussion of these results follows.

Role of NADPH Oxidase in MV-induced Oxidative Stress

Our first hypothesis for the current experiment was that NADPH oxidase activity

is increased in the diaphragm during MV and contributes to diaphragmatic oxidative

injury and contractile dysfunction. This postulate was based on data from our laboratory

indicating that prolonged MV promotes an increase in total calcium levels (unpublished

observations) and activation of calcium-activated neutral proteases (calpain) in the

diaphragm (10). Since NADPH oxidase is a calcium-activated enzyme, we postulated

that increases in diaphragmatic free calcium could up-regulate NADPH oxidase activity.

Nonetheless, our data do not support the postulate that MV is associated with an increase

in NADPH oxidase activity in the diaphragm following MV.









It is important to note that NADPH oxidase exists in two isoforms and that

reactive oxygen species (ROS) can be produced by both phagocytic and non-phagocytic

isoforms of NADPH oxidase (63). A previous investigation from our laboratory has

shown that MV is not associated with an increase in phagocytic cells (35). These

previous findings agree with the current investigation in that MV did not result in an

increase in myeloperoxidase (MPO) activity in the diaphragm; MPO is considered to be

an excellent marker of neutrophil infiltration (62). Therefore, based on these collective

findings, we conclude that phagocytic NADPH oxidase activity does not increase and

apparently does not contribute to MV-induced diaphragmatic dysfunction.

The non-phagocytic NADPH oxidase found in skeletal muscle is similar to the

phagocytic isoform, however some differences do exist. The phagocytic isoform of

NADPH oxidase is composed of three cytosolic subunits (p47phox, p67phox, and p40phox)

and a membrane-spanning cytochrome bsss composed of two subunits (p22phox and

gp91phox). Upon activation, there is a migration of the cytosolic subunits to the

membrane-spanning cytochrome. In comparison, a recent investigation by Javesghani et

al. has characterized the non-phagocytic NADPH oxidase enzyme complex in rat skeletal

muscle (22). They determined that only four of the five subunits exist in skeletal muscle

(p22phox, gp91phox, p47phox, and p67phox) and they are all constitutively expressed on the

membrane. Therefore, non-phagocytic NADPH oxidase activation does not require the

translocation of subunits from the cytosol to the membrane. Interestingly, the mechanism

by which apocynin inhibits phagocytic NADPH oxidase activity is most likely due to the

inhibition of the enzyme assembly by blocking sulfhydryl groups (23). This could









explain, at least in part, why there was no inhibitory effect of apocynin on skeletal muscle

NADPH oxidase activity in the current experiments (table 2).

Our decision to use apocynin as an NADPH oxidase inhibitor was based on

previous investigations showing its inhibitory effect on NADPH oxidase in skeletal

muscle (22, 24). However, during the course of the current experiment, a new report

appeared in the literature suggesting that apocynin has limited efficacy as a non-

phagocytic NADPH oxidase inhibitor (64). This report revealed that the in vitro addition

of apocynin inhibited ROS formation in phagocytic cells (i.e., macrophages) but was not

effective in the inhibition of NADPH oxidase activity in non-phagocytic cells (i.e.,

vascular fibroblasts).

If apocynin does not inhibit NADPH oxidase activity in skeletal muscle, why does

the administration of apocynin prevent MV-induced diaphragmatic contractile

dysfunction? A definitive answer to this question is unavailable but it is possible that

apocynin acts as an intracellular antioxidant to retard MV-induced oxidative stress and

protect against oxidant-mediated diaphragmatic contractile dysfunction. Indeed, the

molecular structure of apocynin contains a phenol group with oxidant scavenging

capacity. The likelihood that apocynin is a physiological antioxidant could explain our

Ending that treatment with apocynin prevented both MV-induced protein oxidation and

diaphragmatic contractile dysfunction. The possibility that apocynin is a physiologically

useful antioxidant warrants further investigation to determine if this compound is a

clinically useful countermeasure to prevent MV-induced diaphragmatic oxidative stress

and contractile dysfunction.









Role of Nitric Oxide Synthase (NOS) in MV-induced Oxidative Stress

The second hypothesis tested in this investigation was that NOS levels are

elevated during MV, increasing the nitration of diaphragmatic proteins. This postulate

was formulated from our preliminary data indicating that MV results in an increase in

total calcium in the diaphragm and knowledge that both of the constitutive isoforms of

NOS (eNOS and nNOS) are calcium-activated enzymes that produce NO. Therefore, an

MV-induced increase in calcium could activate one or both of these NOS isoforms

leading to the formation of NO and the nitration (e.g., 3-nitrotyrosine) of diaphragmatic

proteins. Nonetheless, our results revealed that 18 hours of MV was not associated with

an increase in diaphragmatic levels NOS or 3-nitrotyrosine.

Our finding that MV-induced inactivity in the diaphragm does not alter the levels

of any NOS isoform differs from a previous report by Nguyen and Tidball indicating that

nNOS levels decrease in mice locomotor skeletal muscle following 10 days of hindlimb

unloading (41). Changes in muscle levels of nNOS could be physiologically significant

because an increase in NO in inactive skeletal muscle can be beneficial by inhibiting

calpain and, therefore, reducing the degradation of cytoskeletal proteins such as talin and

vinculin (65). Note, however, although no changes in nNOS levels occurred during 18

hours of MV in the current study, longer periods of MV may decrease levels of nNOS

leaving the diaphragm more susceptible to degradation by calpain.

The finding that MV is not associated with an increase in the levels of 3-

nitrotyrosine in the diaphragm suggests that NO production was not accelerated in the

diaphragm during MV. Our analysis of 3-nitrotyrosine levels in the diaphragm was

comprehensive and evaluated the nitration of proteins in a wide variety of protein pools

in the cell including the insoluble, cytosolic, mitochondrial, and membrane protein









fractions of diaphragm fibers following MV. This type of comprehensive analysis is

important to detect small treatment-induced changes in 3-nitrotyrosine because a previous

investigation by Barreiro et al. has revealed that the nitration of muscle proteins can be

limited to one or two protein compartments of the muscle fiber (58). For example, these

investigators determined that sepsis-induced nitration of proteins was limited to the

membrane and mitochondrial fractions of diaphragm (58). Therefore, the failure to

separate muscle proteins into sub-fractions could mask increases in nitration within small

pools of protein within the fiber. However, based upon our comprehensive analysis of 3-

nitroyrosine in the diaphragm, the current results indicate that 18 hours of MV is not

associated with an increase in the nitration of diaphragmatic proteins.

Critique of Experimental Model

Obtaining a diaphragm muscle biopsy is invasive; therefore, a non-human model

must be utilized to study the effects of MV on the diaphragm. The current investigation

utilized the rat as the experimental model for two reasons: 1) the animal size permits the

necessary surgical techniques to be conducted and also allows for the removal of several

arterial blood samples which is necessary for the maintenance of blood gas homeostasis,

and more importantly, 2) human and rat diaphragms are similar anatomically and

functionally as well as having similar fiber type composition (7, 8).

Controlled MV was utilized in the current study versus pressure-assist MV.

Complete inactivity of the diaphragm results from controlled MV and is utilized

clinically in cases of drug overdose, spinal cord injury, surgery and is also common in

pediatric patients (3).

Sodium pentobarbital was administered as the anesthetic because the current and

previous investigations clearly indicate that sodium pentobarbital does not compromise









the function of the diaphragm (4, 10, 21). Apocynin was utilized in the current

investigation because previous studies have reported that it is an effective inhibitor of

NADPH oxidase in skeletal muscle (22, 24). Another common inhibitor of NADPH

oxidase, diphenylene iodonium (DPI), was not utilized in these experiments because this

compound has been shown to be non-selective for NADPH oxidase. Indeed, DPI has

been shown to inhibit multiple flavoproteins (66) including NADPH oxidase (67, 68),

NOS (69), xanthine oxidase (70), mitochondrial NADH-ubiquinone oxidoreductase (71,

72), and cytochrome p450 (73).

Conclusions

These are the first experiments to investigate the sources of oxidant production in

the diaphragm during prolonged MV. Our studies reveal that MV-induced diaphragmatic

dysfunction and oxidative stress is not due to an increase in NADPH oxidase activity.

Moreover, the in vivo administration of apocynin attenuates the diaphragmatic contractile

dysfunction and oxidative stress induced by MV via its antioxidant properties.

Furthermore, MV does not promote an increase in any of the NOS isoforms, NOS

activity or cause the nitration of proteins in the diaphragm. Therefore, it can be concluded

that neither NADPH oxidase nor NOS contributes to diaphragmatic contractile

dysfunction and oxidative stress that occurs during MV.

Future Directions

The current investigation provides evidence that NADPH oxidase and NOS are

not involved in MV-induced diaphragmatic oxidant stress and contractile dysfunction.

However, several other sources of oxidants exist in skeletal muscle fibers. For example,

the mitochondria can produce superoxide at complex I and III along the electron transport

chain (44). Although the investigation of mitochondrial oxidants in the diaphragm during









prolonged mechanical ventilation is complicated, use of mitochondrial targeted

antioxidants could be a useful probe to study this phenomenon.

Moreover, increases in calcium activated neutral proteases (calpain) can up-

regulate xanthine oxidase activity resulting in the formation of excess superoxide

production (44). Our laboratory has reported an increase in calpain activity during MV

(10). Therefore, the increase in calpain activity seen during MV may lead to the

production of superoxide via xanthine oxidase. Future experiments using specific

pharmacological probes to inhibit xanthine oxidase activity could prove useful in

investigating the role of this oxidant producing pathway.

Finally, skeletal muscle atrophy is associated with an increase in free, "reactive"

iron (48). Free iron can react with hydrogen peroxide and superoxide to produce the

highly reactive hydroxyl radical (44). Therefore, it is possible that MV-induced increases

in free iron could be a source of reactive oxygen species in the diaphragm (10). A variety

of membrane permeable iron chelating compounds exist and be useful in investigating the

role that reactive iron plays in MV-induced oxidant production in the diaphragm.

Determining which of the potential sources of oxidant production is involved in MV-

induced oxidative stress is important and could provide a therapeutic intervention to

retard MV-induced diaphragmatic contractile dysfunction.
















LIST OF REFERENCES


1. Lemaire F. Difficult weaning. Intensive Care M~ed 1993;19 Suppl 2:S69-73.

2. Esteban A, Alia I, Ibanez J, Benito S, Tobin MJ. Modes of mechanical ventilation
and weaning. A national survey of Spanish hospitals. The Spanish Lung Failure
Collaborative Group. Chest 1994;106: 1188-93.

3. Hess D, Kacmarek R. 1996. Essentials of mechanical ventilation. McGraw-Hill,
New York.

4. Powers SK, Shanely RA, Coombes JS, Koesterer TJ, McKenzie M, Van
Gammeren D, Cicale M, Dodd SL. Mechanical ventilation results in progressive
contractile dysfunction in the diaphragm. JAppl Physiol 2002;92: 1851-8.

5. Sassoon CS, Zhu E, Caiozzo VJ. Assist-control mechanical ventilation attenuates
ventilator-induced diaphragmatic dysfunction. Am JRespir Crit Calre M~ed
2004;170:626-32.

6. Metzger JM, Scheidt KB, Fitts RH. Histochemical and physiological characteristics
of the rat diaphragm. JAppl Physiol 1985;58:1085-91.

7. Mizuno M. Human respiratory muscles: fibre morphology and capillary supply.
Eur Respir J 1991;4:587-601.

8. Powers SK, Demirel HA, Coombes JS, Fletcher L, Calliaud C, Vrabas I, Prezant D.
Myosin phenotype and bioenergetic characteristics of rat respiratory muscles. M~ed
Sci Sports Exerc 1997;29: 1573-9.

9. Le Bourdelles G, Viires N, Boczkowski J, Seta N, Pavlovic D, Aubier M. Effects
of mechanical ventilation on diaphragmatic contractile properties in rats. Am J
Respir Crit Care M~ed 1994; 149: 153 9-44.

10. Shanely RA, Zergeroglu MA, Lennon SL, Sugiura T, Yimlamai T, Enns D,
Belcastro A, Powers SK. Mechanical ventilation-induced diaphragmatic atrophy is
associated with oxidative injury and increased proteolytic activity. Am JRespir Crit
Care M~ed 2002; 166: 13 69-74.

11. Anzueto A, Peters JI, Tobin MJ, de los Santos R, Seidenfeld JJ, Moore G, Cox WJ,
Coalson JJ. Effects of prolonged controlled mechanical ventilation on
diaphragmatic function in healthy adult baboons. Crit Care M~ed 1997;25:1 187-90.









12. Yang L, Luo J, Bourdon J, Lin MC, Gottfried SB, Petrof BJ. Controlled mechanical
ventilation leads to remodeling of the rat diaphragm. Am JRespir Crit Calre M~ed
2002;166:1135-40.

13. Gayan-Ramirez G, de Paepe K, Cadot P, Decramer M. Detrimental effects of short-
term mechanical ventilation on diaphragm function and IGF-I mRNA in rats.
Intensive Care M~ed 2003;29:825-33.

14. Bernard N, Matecki S, Py G, Lopez S, Mercier J, Capdevila X. Effects of
prolonged mechanical ventilation on respiratory muscle ultrastructure and
mitochondrial respiration in rabbits. Intensive Care M~ed 2003;29: 1 11-8.

15. Zergeroglu MA, McKenzie MJ, Shanely RA, Van Gammeren D, DeRuisseau KC,
Powers SK. Mechanical ventilation-induced oxidative stress in the diaphragm. J
Appl Physiol 2003;95:1116-24.

16. Dean RT, Fu S, Stocker R, Davies MJ. Biochemistry and pathology of radical-
mediated protein oxidation. Biochem J 1997;324 (Pt 1): 1-18.

17. Shringarpure R, Davies KJ. Protein turnover by the proteasome in aging and
disease. Free RadiRRR~~~~~RRRRR~~~~c Biol 2ed 2002;32: 1084-9.

18. Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method
for carbonyl assay. Methods Enzymol 1994;233:357-63.

19. Reid MB. Invited Review: redox modulation of skeletal muscle contraction: what
we know and what we don't. JAppl Physiol 2001;90:724-31.

20. Powers SK, Demirel HA, Vincent HK, Coombes JS, Naito H, Hamilton KL,
Shanely RA, Jessup J. Exercise training improves myocardial tolerance to in vivo
ischemia-reperfusion in the rat. Am JPhysiol 1998;275:R1468-77.

21. Betters J, Criswell DS, Shanely RA, Van Gammeren D, Falk DJ, DeRuisseau KC,
Deering M, Yimlamai T, Powers SK. Trolox attenuates mechanical ventilation-
induced diaphragmatic dysfunction and proteolysis. Am JRespir Crit Calre M~ed
2004;170:1179-1184.

22. Javesghani D, Magder SA, Barreiro E, Quinn MT, Hussain SN. Molecular
characterization of a superoxide-generating NAD(P)H oxidase in the ventilatory
muscles. Am J Respir Crit Care M~ed 2002; 165:412-8.

23. Simons JM, Hart BA, Ip Vai Ching TR, Van Dijk H, Labadie RP. Metabolic
activation of natural phenols into selective oxidative burst agonists by activated
human neutrophils. Free Radic Biol2~ed 1990;8:251-8.

24. Supinski G, Stofan D, Nethery D, Szweda L, DiMarco A. Apocynin improves
diaphragmatic function after endotoxin administration. JAppl Physiol
1999;87:776-82.









25. Lawler JM, Song W. Specificity of antioxidant enzyme inhibition in skeletal
muscle to reactive nitrogen species donors. Biochem Biophys Res Commun
2002;294:1093-100.

26. Criswell DS, Shanely RA, Betters JJ, McKenzie MJ, Sellman JE, Van Gammeren
DL, Powers SK. Cumulative effects of aging and mechanical ventilation on in vitro
diaphragm function. Chest 2003;124:2302-8.

27. Radell PJ, Remahl S, Nichols DG, Eriksson LI. Effects of prolonged mechanical
ventilation and inactivity on piglet diaphragm function. Intensive Calre M~ed
2002;28:358-64.

28. Sassoon CS, Caiozzo VJ, Manka A, Sieck GC. Altered diaphragm contractile
properties with controlled mechanical ventilation. JAppl Physiol 2002;92:2585-95.

29. Watson AC, Hughes PD, Louise Harris M, Hart N, Ware RJ, Wendon J, Green M,
Moxham J. Measurement of twitch transdiaphragmatic, esophageal, and
endotracheal tube pressure with bilateral anterolateral magnetic phrenic nerve
stimulation in patients in the intensive care unit. Crit Care M~ed 2001;29: 1325-31.

30. Gayan-Ramirez G, Decramer M. Effects of mechanical ventilation on diaphragm
function and biology. Eur Respir J 2002;20: 1579-86.

31. Racz GZ, Gayan-Ramirez G, Testelmans D, Cadot P, De Paepe K, Zador E,
Wuytack F, Decramer M. Early changes in rat diaphragm biology with mechanical
ventilation. Am J Respir Crit Care M~ed 2003;168:297-3 04.

32. Capdevila X, Lopez S, Bernard N, Rabischong E, Ramonatxo M, Martinazzo G,
Prefaut C. Effects of controlled mechanical ventilation on respiratory muscle
contractile properties in rabbits. Intensive Care M~ed 2003;29: 103-10.

33. Shanely RA, Coombes JS, Zergeroglu AM, Webb AI, Powers SK. Short-duration
mechanical ventilation enhances diaphragmatic fatigue resistance but impairs force
production. Chest 2003;123:195-201.

34. Shanely RA, Van Gammeren D, Deruisseau KC, Zergeroglu AM, McKenzie MJ,
Yarasheski KE, Powers SK. Mechanical ventilation depresses protein synthesis in
the rat diaphragm. Am JRespir Crit Care M~ed 2004; 170:994-9.

35. Van Gammeren D, Falk DJ, DeRuisseau KC, Sellman JE, Decramer M, Powers
SK. Reloading the diaphragm following mechanical ventilation does not promote
injury. Chest 2005;127:2204-2210.

36. Vassilakopoulos T, Petrof BJ. Ventilator-induced diaphragmatic dysfunction. Am J
Respir Crit Care M~ed 2004; 169:336-41.

37. Knisely AS, Leal SM, Singer DB. Abnormalities of diaphragmatic muscle in
neonates with ventilated lungs. J Pediatr 1988; 113:1074-7.










38. Ku Z, Yang J, Menon V, Thomason DB. Decreased polysomal HSP-70 may slow
polypeptide elongation during skeletal muscle atrophy. Am JPhysiol
1995;268:C1369-74.

39. Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA, Poueymirou
WT, Panaro FJ, Na E, Dharmaraj an K, Pan ZQ, Valenzuela DM, DeChiara TM,
Stitt TN, Yancopoulos GD, Glass DJ. Identification of ubiquitin ligases required
for skeletal muscle atrophy. Science 2001;294:1704-8.

40. Kandarian SC, Stevenson EJ. Molecular events in skeletal muscle during disuse
atrophy. Exerc Sport Sci Rev 2002;30: 111-6.

41. Nguyen HX, Tidball JG. Expression of a muscle-specific, nitric oxide synthase
transgene prevents muscle membrane injury and reduces muscle inflammation
during modified muscle use in mice. JPhysiol 2003;550:347-56.

42. Turrens JF. Superoxide production by the mitochondrial respiratory chain. Biosci
Rep 1997; 17:3-8.

43. Boveris A, Chance B. The mitochondrial generation of hydrogen peroxide. General
properties and effect of hyperbaric oxygen. Biochent J 1973;134:707-16.

44. Halliwell B, Gutteridge J. 1999. Free radicals in biology and medicine, 3 ed.
Oxford University Press, New York.

45. Kondo H. 2000. Oxidative stress in muscular atrophy. Dr C. Sen, L. Packer and O.
Hanninen, editors. Handbook of oxidants and antioxidants in exercise. Elsevier
Science, St Louis.

46. Kondo H, Kimura M, Itokawa Y. Manganese, copper, zinc, and iron concentrations
and subcellular distribution in two types of skeletal muscle. Proc Soc Exp Biol2~ed
1991;196:83-8.

47. Gilbert HF. Biological disulfides: the third messenger? Modulation of
phosphofructokinase activity by thiol/disulfide exchange. JBiol Chent
1982;257:12086-91.

48. Kondo H, Miura M, Nakagaki I, Sasaki S, Itokawa Y. Trace element movement
and oxidative stress in skeletal muscle atrophied by immobilization. Am JPhysiol
1992;262:E583-90.

49. Ryter SW, Tyrrell RM. The heme synthesis and degradation pathways: role in
oxidant sensitivity. Heme oxygenase has both pro- and antioxidant properties. Free
Radic Biol 2ed 2000;28:28 9-3 09.

50. Kondo H, Nakagaki I, Sasaki S, Hori S, Itokawa Y. Mechanism of oxidative stress
in skeletal muscle atrophied by immobilization. Am JPhysiol 1993;265:E83 9-44.









51. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. NEnglJ
M~ed 1985;312: 159-63.

52. Booth FW, Giannetta CL. Effect of hindlimb immobilization upon skeleton muscle
calcium in rat. Calcif Tissue Res 1973;13:327-30.

53. DeLeo FR, Quinn MT. Assembly of the phagocyte NADPH oxidase: molecular
interaction of oxidase proteins. JLeukoc Biol 1996;60:677-91.

54. Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in
cardiovascular biology and disease. Circ Res 2000;86:494-501.

55. Reid MB. Role of nitric oxide in skeletal muscle: synthesis, distribution and
functional importance. Acta Physiol Scand 1998; 162:401-9.

56. Stamler JS, Singel DJ, Loscalzo J. Biochemistry of nitric oxide and its redox-
activated forms. Science 1992;258:1898-902.

57. Freeman B. Free radical chemistry of nitric oxide. Looking at the dark side. Chest
1994;105:79S-84S.

58. Barreiro E, Comtois AS, Gea J, Laubach VE, Hussain SN. Protein tyrosine
nitration in the ventilatory muscles: role of nitric oxide synthases. Am JRespir Cell
M~olBiol 2002;26:43 8-46.

59. Segal SS, White TP, Faulkner JA. Architecture, composition, and contractile
properties of rat soleus muscle grafts. Am JPhysiol 1986;250:C474-9.

60. Cui XL, Douglas JG. Arachidonic acid activates c-jun N-terminal kinase through
NADPH oxidase in rabbit proximal tubular epithelial cells. Proc NatlAcad Sci U S
A 1997;94:3771-6.

61. Bradford MM. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem
1976;72:248-54.

62. Seekamp A, Mulligan MS, Till GO, Ward PA. Requirements for neutrophil
products and L-arginine in ischemia-reperfusion injury. Am JPathol
1993;142:1217-26.

63. Bokoch GM, Knaus UG. NADPH oxidases: not just for leukocytes anymore!i
Trends Biochem Sci 2003;28:502-8.

64. Vejrazka M, Micek R, Stipek S. Apocynin inhibits NADPH oxidase in phagocytes
but stimulates ROS production in non-phagocytic cells. Biochim Biophys Acta
2005; 1722:143-7.










65. Koh TJ, Tidball JG. Nitric oxide inhibits calpain-mediated proteolysis of talin in
skeletal muscle cells. Am JPhysiol Cell Physiol 2000;279:C806-12.

66. Abdelrahman M, Mazzon E, Bauer M, Bauer I, Delbosc S, Cristol JP, Patel NS,
Cuzzocrea S, Thiemermann C. Inhibitors of NADPH oxidase reduce the organ
injury in hemorrhagic shock. Shock 2005;23:107-14.

67. O'Donnell BV, Tew DG, Jones OT, England PJ. Studies on the inhibitory
mechanism of iodonium compounds with special reference to neutrophil NADPH
oxidase. Biochent J 1993;290 ( Pt 1):41-9.

68. Cross AR, Jones OT. The effect of the inhibitor diphenylene iodonium on the
superoxide-generating system of neutrophils. Specific labelling of a component
polypeptide of the oxidase. Biochent J 1986;237: 1 11-6.

69. Stuehr DJ, Fasehun OA, Kwon NS, Gross SS, Gonzalez JA, Levi R, Nathan CF.
Inhibition of macrophage and endothelial cell nitric oxide synthase by
diphenyleneiodonium and its analogs. Fa~seb J 1991;5:98-103.

70. Zhang Z, Blake DR, Stevens CR, Kanczler JM, Winyard PG, Symons MC,
Benboubetra M, Harrison R. A reappraisal of xanthine dehydrogenase and oxidase
in hypoxic reperfusion injury: the role of NADH as an electron donor. Free Radic
Res 1998;28:151-64.

71. Ragan CI, Bloxham DP. Specific labelling of a constituent polypeptide of bovine
heart mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone
reductase by the inhibitor diphenyleneiodonium. Biochent J1977; 163:605-15.

72. Li Y, Trush MA. Diphenyleneiodonium, an NAD(P)H oxidase inhibitor, also
potently inhibits mitochondrial reactive oxygen species production. Biochent
Biophys Res Conanun 1998;253:295-9.

73. Chen L, Yu LJ, Waxman DJ. Potentiation of cytochrome P450/cyclophosphamide-
based cancer gene therapy by coexpression of the P450 reductase gene. Cancer Res
1997;57:4830-7.
















BIOGRAPHICAL SKETCH

Darin Lee Van Gammeren was born in Luverne, Minnesota, and raised on a farm in

rural Inwood, Iowa. He graduated from the University of Sioux Falls, Sioux Falls, South

Dakota, with a bachelor' s degree in exercise science. In 1999 he moved to Kearney,

Nebraska, where he attended the University of Nebraska at Kearney and received a

master' s degree in exercise science. Darin began work on a Doctor of Philosophy degree

in exercise physiology in 2001 at the University of Florida. Darin will begin post-

doctoral training at Boston University in the area of skeletal muscle disuse atrophy under

the direction of Dr. Susan Kandarian in July 2005.