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Enhanced Degradation of Oxidatively Modified Myofibrillar Proteins by Calpain and Caspase-3

Permanent Link: http://ufdc.ufl.edu/UFE0022699/00001

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Title: Enhanced Degradation of Oxidatively Modified Myofibrillar Proteins by Calpain and Caspase-3
Physical Description: 1 online resource (52 p.)
Language: english
Creator: Smuder, Ashley
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Applied Physiology and Kinesiology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Prolonged periods of skeletal muscle inactivity results in fiber atrophy. The physiological consequences of disuse-induced muscle atrophy is a diminished muscle force generating capacity which could negatively impact activities of daily living. Importantly, oxidative stress has been linked to the signaling events responsible for protein degradation. However, the mechanisms by which oxidative stress increases the rate of proteolysis have not been fully elucidated. In theory, oxidation of proteins can alter their structure to affect susceptibility to proteolytic processing. Several proteolytic systems are capable of degrading muscle proteins. Calpain (I and II) and caspase-3 are proteases that are capable of degrading intact myofilament proteins and are also activated during disuse atrophy. Therefore, we hypothesized that oxidative modification of skeletal muscle proteins increases their susceptibility to degradation by calpain and/or caspase-3. To test this postulate we introduced isolated myofibrillar proteins from the diaphragm to four distinct levels of oxidation and subsequently treated each group independently with active calpain I, calpain II, and caspase-3. Protein degradation was measured using protein mapping. Our findings reveal, as oxidation of myofibrillar proteins increases, their degradation by calpain (I and II) and caspase-3 increases as well. Therefore, we conclude that oxidative modification of myofibrillar proteins accelerates protein degradation by both calpains and caspase-3.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ashley Smuder.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Powers, Scott K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022699:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022699/00001

Material Information

Title: Enhanced Degradation of Oxidatively Modified Myofibrillar Proteins by Calpain and Caspase-3
Physical Description: 1 online resource (52 p.)
Language: english
Creator: Smuder, Ashley
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Applied Physiology and Kinesiology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Prolonged periods of skeletal muscle inactivity results in fiber atrophy. The physiological consequences of disuse-induced muscle atrophy is a diminished muscle force generating capacity which could negatively impact activities of daily living. Importantly, oxidative stress has been linked to the signaling events responsible for protein degradation. However, the mechanisms by which oxidative stress increases the rate of proteolysis have not been fully elucidated. In theory, oxidation of proteins can alter their structure to affect susceptibility to proteolytic processing. Several proteolytic systems are capable of degrading muscle proteins. Calpain (I and II) and caspase-3 are proteases that are capable of degrading intact myofilament proteins and are also activated during disuse atrophy. Therefore, we hypothesized that oxidative modification of skeletal muscle proteins increases their susceptibility to degradation by calpain and/or caspase-3. To test this postulate we introduced isolated myofibrillar proteins from the diaphragm to four distinct levels of oxidation and subsequently treated each group independently with active calpain I, calpain II, and caspase-3. Protein degradation was measured using protein mapping. Our findings reveal, as oxidation of myofibrillar proteins increases, their degradation by calpain (I and II) and caspase-3 increases as well. Therefore, we conclude that oxidative modification of myofibrillar proteins accelerates protein degradation by both calpains and caspase-3.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ashley Smuder.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Powers, Scott K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022699:00001


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ENHANCED DEGRADATION OF OXIDATI VELY MODIFIED MYOFIBRILLAR PROTEINS BY CALPAIN AND CASPASE-3 By ASHLEY JOSLIN SMUDER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008 1

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2008 Ashley Joslin Smuder 2

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To my family and friends for their constant support and to the people who have played a significant role in my education 3

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ACKNOWLEDGMENTS First, I thank my mentor (Dr. Scott Powe rs) for his guidance and continuous support. Also, I commend my thesis committee members (D r. Stephen Dodd and Dr. David Criswell) for their direction and support. Of course I wish to thank all laboratory members who played a role in my achievements. Finally, I am thankful for my family and friends who share my life and career experiences. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF FIGURES .........................................................................................................................7 ABSTRACT ...................................................................................................................... ...............8 1 INTRODUCTION ................................................................................................................ ....9 2 LITERATURE REVIEW .......................................................................................................11 Overview of Skeletal Muscle Disuse Atrophy .......................................................................11 Introduction .................................................................................................................. ...11 Skeletal Muscle Characteristics .......................................................................................12 Mechanisms of Skeletal Muscle Dysfunction .................................................................12 Protein Synthesis and Degradation ..................................................................................12 Proteolytic Systems .........................................................................................................13 Oxidative Stress and Disuse Muscle Atrophy .................................................................16 Summary ....................................................................................................................... ..........18 3 MATERIALS AND METHODS ...........................................................................................21 Experiment 1: Animals ......................................................................................................... ..21 Animal Model Justification .............................................................................................21 Animal Housing and Diet ................................................................................................21 Experimental Design .......................................................................................................21 Animal Protocol ...............................................................................................................22 Statistical Analysis .......................................................................................................... 22 General Methods .....................................................................................................................22 Biochemical Measurements ....................................................................................................22 4 RESULTS ..................................................................................................................... ..........26 Redox Balance ........................................................................................................................26 Myofibril Digestion ................................................................................................................26 Identification of Specifi c Protein Substrates ..........................................................................28 5 DISCUSSION .................................................................................................................. .......40 Overview of Principal Findings ..............................................................................................40 Calpains and Caspase-3 Release Myofilaments ..............................................................40 Induction of Oxidative Stress during Disuse Atrophy ....................................................41 Oxidation of Myofibrils Increases Protei n Degradation by Calpain and Caspase-3 .......41 Conclusions and Future Directions .........................................................................................42 5

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LIST OF REFERENCES ...............................................................................................................44 BIOGRAPHICAL SKETCH .........................................................................................................52 6

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LIST OF FIGURES Figure page 2-1 Pathway of protein degradation by calpain followed by degradation by the proteasome. ................................................................................................................... .....19 2-2 Potential pathways for the act ivation of caspase-3 during MV ........................................20 3-1 Experimental model for examining the effects of various levels of oxidation on isolated diaphragm myofibrilla r proteins on the activation of caspase-3 and calpain. ......25 4-1 Determination of oxidative damage ...................................................................................30 4-2 Isolated myofilaments from diaphrag m muscle .................................................................31 4-3 250 kDa band intensity (percen t different versus control) .................................................32 4-4 100 kDa band intensity (percen t different versus control) .................................................33 4-5 37 kDa band intensity (percen t different versus control) ...................................................34 4-6 30 kDa band intensity (percen t different versus control) ...................................................35 4-7 Myosin protein (percent different versus control) .............................................................36 4-8 Actin protein (percent different versus control) .................................................................37 4-9 -actinin protein (percent di fferent versus control) ...........................................................38 4-10 Troponin protien (percent di fferent versus control) ...........................................................39 7

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8 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INCREASED DEGRADATION OF OXIDATI VELY MODIFIED MYOFIBRILLAR PROTEINS BY CALPAIN AND CASPASE-3 By Ashley Joslin Smuder December 2008 Chair: Scott K. Powers Major: Applied Physio logy and Kinesiology Prolonged periods of skeletal muscle inactiv ity results in fiber atrophy. The physiological consequences of disuse-induced muscle atrophy is a diminished muscle force generating capacity which could negatively impact activities of dail y living. Importantly, oxidative stress has been linked to the signaling events responsible for pr otein degradation. However, the mechanisms by which oxidative stress increases the rate of proteolysis have not b een fully elucidated. In theory, oxidation of proteins can alter th eir structure to affect susceptib ility to proteolytic processing. Several proteolytic systems are capable of degr ading muscle proteins. Calpain (I and II) and caspase-3 are proteases that are capable of degr ading intact myofilament proteins and are also activated during disuse atrophy. Therefore, we hypothesized that oxidative modification of skeletal muscle proteins increases their suscepti bility to degradation by calpain and/or caspase-3. To test this postulate we introduced isolated myofibrillar proteins from the diaphragm to four distinct levels of oxidation a nd subsequently treated each group independently with active calpain I, calpain II, and caspa se-3. Protein degradation was measured using protein mapping. Our findings reveal, as oxidation of myofibrillar proteins increases, their degradation by calpain (I and II) and caspase-3 increases as well. Theref ore, we conclude that oxidative modification of myofibrillar proteins accelerates protein degradation by both calpains and caspase-3.

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CHAPTER 1 INTRODUCTION Skeletal muscle inactivity produces a profound atrophy of muscle fibe rs. This atrophy is characterized by a decrease in fiber size and ther efore a decrease in muscle mass (29). Bed rest, immobilization, spaceflight, muscle denervation, a nd mechanical ventilation are all conditions that promote skeletal muscle atrophy. Locomo tor muscles affected in these conditions are normally involved in maintaining posture (15). Loss of functional capacity in these muscles can greatly impact daily life therefore understandi ng the mechanisms that contribute to disuse atrophy is imperative. Skeletal muscle disuse results in an imbalance in muscle protein synthesis and degradation. During this catabo lic condition there is a decrease in muscle protein synthesis followed by an increase in protein degradation. The proteolytic pathways involved in protein degradation are well known. Majo r proteolytic systems in skeletal muscle include the ubiquitinproteasome system, calpains, and caspases. Importa ntly, these pathways appear to be markedly influenced by oxidative stress. To date, all forms of disuse muscle atrophy are associated with an increase in protein oxidation. This is significant because oxidative modification of mu scle proteins during disuse could increase their susceptibility to degradati on. However, the mechanisms by which oxidative stress influences the rate of proteolysis are lacking (1, 46, 69, 71, 76, 82). In theory, oxidation of proteins can alter their structur e to affect susceptibility to proteolytic processing (21, 22). Evidence indicates that protei n oxidation enhances substrate recognition for the proteasome; however, this system is unable to degrade intact sarcomeric proteins. Calpain (I and II) and caspase-3 are proteases that are capable of degrading intact myofilament proteins and are also activated dur ing disuse atrophy. Theref ore, these experiments 9

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10 determined whether oxidative modification of myofib rillar proteins increase s their susceptibility to degradation via calpain (I and II) or caspase3. Specifically, our experi ments were designed to achieve the following specific aim. Specific Aim: To determine if oxidation influences myofibrillar prot ein breakdown when muscle proteins are independently exposed to active calpain I, calpain II, and caspase-3. Rationale: Our previous work has demonstrated that inactivity-induced muscle atrophy is associated with oxidative stress and that the induction of oxidativ e stress accelerates proteolytic activity. Curr ently, the influence of muscle prot ein oxidation on substrate recognition for calpain and caspase-3 is unknown. Hypothesis: Oxidative modification of myofibri llar proteins will increase their vulnerability to degradation by both calpain (I and II) and caspase-3.

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CHAPTER 2 LITERATURE REVIEW Periods of skeletal muscle disuse result in contractile dysfunction due to both a loss of muscle mass and a decrease in specific force generating capacity. Evidence suggests that oxidative stress is an important regulator in the pathways l eading to muscle atrophy during periods of disuse. Models of disuse include: i mmobilization, chronic bed rest, physical inactivity, spaceflight, hindlimb unloading, and mechanical vent ilation. These models are studied in order to better understand the mechanisms that contri bute to muscle atrophy during disuse so that protective countermeasures can be developed. Skeletal muscle weakness due to atrophy and contractile dysf unction can be attributed to changes in the rate of proteoly sis (64, 76). Therefore, the first segment of this chapter will discuss the mechanisms of skeletal muscle at rophy and outline the different proteolytic systems involved in disuse-induced atrophy. The second segment of this chap ter will discuss the induction of oxidative stress duri ng inactivity. More sp ecifically, oxidative damage occurs in skeletal muscle during periods of disuse and the second segment of this chapter will address both the mechanisms responsible for oxidative stress a nd the contribution of oxidative modification of proteins to skeletal muscle weakness. Overview of Skeletal Muscle Disuse Atrophy Introduction Skeletal muscle tissue consis ts of ~40% of total body mass and provides basic functions such as locomotion, metabolism and respiration. Skeletal muscle exhibits a very high level of plasticity. Skeletal muscle hypertrophy is characterized by increased muscle size, protein content and strength (31, 32). Conversely, prolonged inactiv ity results in the loss of skeletal muscle mass. Skeletal muscle disuse atrophy occurs as a result of altered protein metabolism which 11

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leads to a decrease in muscle contractile protein content and function (26, 80, 81, 88, 90). Due to the severity of complications ar ising from skeletal muscle disuse atrophy identification of the mechanism(s) responsible for inactiv ity-induced weakness is important. Skeletal Muscle Characteristics Skeletal muscle proteins can be divided into three classes based upon their solubility and location in the muscle. Sarcoplasmic proteins cons titute ~30-35% of skeletal muscle and are the cytoplasmic proteins soluble in low salt solutions. These proteins comprise all of the glycolytic enzymes (19, 20). Stroma proteins constitute ~1015% of total protein in muscle and are those proteins that are insoluble in aqueous solvent at neutral pH (1 9, 20). The third and largest class of skeletal muscle proteins are the myofibrilla r proteins which constitute ~55-60% of total muscle protein (19, 20). Myofibrillar proteins consist of the myofib ril or contractile structure in skeletal muscle. They are responsible for the contra ctile properties of muscle and for most of the functional properties (19, 20). Mechanisms of Skeletal Muscle Dysfunction Protein Synthesis and Degradation Skeletal muscle mass is maintained through th e balance of the rate of protein synthesis and protein degradation. Periods of prolonged muscle disuse can result in muscle wasting and alter the muscles physiological function. Thes e conditions have been demonstrated during periods of immobilization, micro-gravity, muscle denervation, and mechan ical ventilation (31, 32, 62, 87). Skeletal muscle atrophy is caused by a decrease in muscle protein synthesis and an increase in muscle protein proteo lysis (31, 32). The decrease in pr otein synthesis is characterized by alterations in translational initiation and elongation and/or a decrease in cellular RNA (28, 41, 58, 59). Our laboratory has observed a decrease in di aphragmatic protein synt hesis in as few as 6 hours following mechanical ventilation (75). Nevertheless, although inactivity results in 12

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decreased protein synthesis our la boratory has also demonstrated that the development of atrophy during periods of inactivity is primarily due to an increase in protein degradation (12, 52, 53, 76). During skeletal muscle proteolysis myofibrillar protei n is lost at a rate faster than other muscle proteins (31). These proteins must first be disassembled before they can be degraded and therefore, release of my ofilaments is a required first step in contractile protein breakdown (19). Proteolytic Systems Disassembly and degradation of skeletal muscle proteins occurs via activation of several proteolytic systems (75, 76). There are at least four different proteoly tic systems involved in skeletal muscle disuse atrophy. These systems include: 1) Lysosomal proteases 2) Ubiquitinproteasome system 3) Ca2+dependent calpain system 4) Caspases. These systems all work together during proteolysis (24, 31, 32, 62, 66). Lysosomal proteases. Lysosomal proteases include the family of proteases called cathepsins. These proteases are found ubiquitously in all tissue, but with great er levels in tissues having higher protein turnover (63) The major role of the cathep sins is to degrade membrane proteins, including receptors, ligands, channels and transporters. Lysosomal proteases are activated during skeletal muscle atrophy and it has been argu ed that these proteases do not appear to significantly affect the rates of my ofibrillar protein degrad ation or total protein degradation (17, 25, 68). Nonetheless, new ev idence questions this concept and suggests lysosomal cathepsins may contribute to muscle protein breakdown via autopagy (47, 96). Hence, determining the precise role that lysosomal proteases play in muscle wasting remains an important area for future research. Ubiquitin-proteasome system. The proteasome is comprised of a 20S core proteasome subunit with a 19S regulatory proteasome subunit attached to each side. The combination of these three subunits makes up the 26S proteas ome complex (21-23, 89). The 26S proteasome 13

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degrades proteins that have been ubiquitinated, wh ereas the 20S is able to degrade proteins that have not been ubiquitinated. The pathway of degr adation by the 26S proteasome complex begins with the protein E1, the ubiquitin-activating en zyme. After ubiquitin is activated it is then transferred to E2, the ubiquitin carrier protein. E2 then interacts with E3, an ubiquitin ligase responsible for catalyzing the tran sfer of ubiquitin to a protein s ubstrate, marking the substrate for proteasomal degradation (46, 70, 85). The 19S regulatory complex is required for degradation of ubiquitinated proteins. Once a protein is ubiquitinated it is subsequently recognized by the 19S regulatory complex. Energy from ATP hydrol ysis removes the polyubiquitin chain and unfolds the substrate protein. The unfolded prot ein then enters the 20S proteasome and is degraded (11, 22, 70). Along with being a part of the 26S proteas ome, the 20S proteasome has also been reported to be located intracellula rly without any regulat ory proteins attached (22). This free 20S proteasome appears to be the predominant in tracellular form. The 20S proteasome is capable of acting separately from ubiquitin and ATP (21, 22). During muscle atrophy, the proteasome system is responsible for the degradation of actin and myosin. However, this system is unable to de grade intact sarcomeres (20, 89) and therefore, these complexes must be broken down by a separa te proteolytic system prior to degradation by the proteasome (89, 93). Ca2+-dependent calpain system. Calpains are calcium-dependent cysteine proteases that are activated in skeletal muscle during conditions that promot e muscle wasting (e.g. inactivity, sepsis, cachexia) (6, 20, 24, 25). When calpains are activated they can cleave myofibrillar proteins and produce large peptides that can later be degraded by the proteasome (17, 30, 68). The calpains refer to cal pain I and calpain II which are also referred to as and m14

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calpain respectively. These names are based upon the amount of calcium each requires to be catalytically active. Skeletal mu scle contains approximately th e same amount of both calpain I and II (20). Calpain (I and II) is located exclusively intracellularly and in the cell most is located on or next to the z-disk of the sarcomere with the I-band and the A-band also containing small amounts (18, 32). Due to the location of calpain the substrates that it prim arily acts on are those involved in linking contractile el ements together, and in general calpain I and II tend to act on the same substrates but at different rates (20). It is unknown if calpains can directly degrade actomyosin complexes but it is clear that calpai n can release sarcomeric proteins by cleaving cytoskeletal proteins that anchor the contractile elements (19, 20). Some of the protein substrates known to be cleaved by calpain activa tion include -spectrin, troponin I, desmin, and titin (34, 47, 68, 89). Due to the damaging effects calpain can have on the integrity of myofibers calpain activity is highly regulated and remains inactive mo st of the time. Calpain activity is regulated by several factors including cytosolic calcium leve ls, phosphorylation, and calpastatin concentration (20). Calpastatin is the only known endogenous inhibitor of calpain. In brief, high cytosolic levels of calpastatin inhibit calpain activation whereas a decrease in calpastatin levels favors calpain activation. Inte restingly, calpastatin is a substrate of the protease caspase-3 and therefore, increased caspase-3 activation can reduce calpasta tin levels and promote calpain activation (20, 61, 62). Caspases. Caspases are cysteine-dependent prot eases that are capable of degrading proteins and of promoting apopt osis (14, 84). Apoptotic pathways are activated in skeletal muscle during disuse atrophy. Apoptotic prot eases have been reported to cleave actin in vitro which suggests that apoptotic proteases contribut e to protein breakdown ( 33, 49). In particular, caspase-3 is reported to not only cleave actin but also degrade actomyosin complexes (55). In the 15

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cell, caspases are expressed as inactive precu rsors called procaspases. Cleavage of the procaspases results in their activa tion, and once a caspase is activated it is able to cleave and activate other caspases (67). This cascade-like activation of caspases can be seen during the activation of caspase-3. Caspase-3 is a protease that has been found to act similarly to calpain in its ability to promote degradati on of cytoskeletal proteins and the release of actin and myosin monomers to be subsequently degraded by the proteasome. Caspase-3 can be activated by a calcium-releasing pathway, a mitochondrial pathway, and calpain is also capable of activating caspase-3 (9, 67). Oxidative Stress and Disuse Muscle Atrophy The relationship between oxidative stress and disuse muscle atrophy was first reported by Kondo (36-39). These studies reporte d that immobilization of skel etal muscles was associated with oxidative injury to muscle. Oxidative stress is important during muscle wasting situations because oxidants play an important role in ma ny signaling pathways th at promote contractile dysfunction, disturbances in calcium homeostasis, and protease activation in skeletal muscle (1, 46, 69, 71, 76, 82, 83). Oxidative stress occurs when th ere is an imbalance in the production of reactive oxidant species (ROS) a nd their ability to be scavenged by antioxidants. Oxidants are able to modify proteins causing them to have a loss of function or a change in function. These changes lead to an enhanced degradation of the oxidized proteins ( 22). Physiologically, the degradation of oxidatively modified proteins is important in order to avoid excessive accumulation of damaged proteins. Therefore degr adation of oxidized and oxidatively modified proteins is an important part of oxidant defense (22). Following oxidation, modifications to amino acids, fragmentation, and aggregation can all be induced. These changes in proteins can ch ange the proteolytic su sceptibility of protein substrates (22). Oxidation of am ino acids can alter protein struct ure to affect susceptibility to 16

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proteolytic processing. Many proteins change their secondary and tertiary structure upon oxidation (4, 22). In order to maintain cellular homeostasis and prevent accumulation of highly oxidized and cross-linked proteins, degradation of oxidized proteins is essential. Intracellular oxidation products need to either be repaired or removed. Increasing eviden ce indicates that cells are able to selectively degrade the oxidized form s of proteins (4). For example, evidence of changes in degradation susceptibility has been re ported for glutamine synthetase. Specifically, it has been reported that oxidative modification of this enzyme increases its susceptibility for degradation by several proteases ( 45, 73). Similar results have b een reported for other oxidized proteins (22). Oxidized proteins in cell free extracts were found to have no ATP dependency during proteolysis. Along with th is, oxidized proteins have been found to be poor substrates for ubiquitination. This means that the 26S proteas ome is not involved in the degradation of oxidatively modified proteins, wh ich then increases their suscepti bility to degradation by the 20S proteasome which does not require ubiquitination or ATP in or der to recognize and degrade proteins (21, 22). Since oxidative stress occurs in skeletal muscle during periods of inactivity and the proteasome system is unable to degrade intact sa rcomeres, the role of calpains and caspase-3 may be important in the disasse mbly of the actomyosin complexe s prior to degradation by the proteasome. Both proteolytic systems have the po ssibility of being activated by oxidative stress, and may also selectively degrade ox idized proteins (40, 44, 62, 67). During oxidative stress, oxidant s generate the formation of reactive aldehydes (i.e. 4HNE). These reactive aldehydes have been shown to reduce plasma membrane Ca2 + ATPase activity (58). This decrease would delay Ca2 + removal from the cell and therefore contribute to cellular Ca2 + accumulation. This overload of Ca2 + in the cell could lead to the activation of 17

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calpain resulting in increased pr oteolysis of diaphragmatic cytoskeletal proteins and the release of myofilaments for subsequent degradation by the proteasome syst em (Figure 2-1). The oxidative increase in Ca2 + overload and the activation of calpa in are also the means by which caspase-3 can be activated. Increases in intracellular Ca2 + concentrations cleave and activate caspase-12 which in turn activates caspase-3. The activation of calpain by increased intracellular Ca2 + concentration also activates caspase-3. Oxid ative stress can also activate caspase-3 via a mitochondrial pathway. Specifically, oxidative stre ss leads to an increas e in the release of cytochrome c from the mitochondria, activating cas pase-9. Activation of cas pase-9 leads to the subsequent activation of caspase-3 enabling the protease to cleave skeletal muscle sarcomeres and release actin and myosin fo r degradation by the proteaso me (Figure 2-2) (ref 9, 67). Summary Inactivity results in skeletal muscle atrophy; this atrophic response to disuse is caused by a decrease in protein synthesis and an increase in degradation. Ou r work reveals that inactivityinduced oxidative stress in respirat ory skeletal muscle is a require ment for proteolytic activation, atrophy, and contractile dysfunction. In this regard, our lab has al so demonstrated that prolonged inactivity results in increased activation of th e proteases, calpain and caspase-3. Calpain (I and II) and caspase-3 can function as the rate-limiting st ep in muscle protein proteolysis by releasing sarcomeres for further degradation by the proteasome. Growing evidence indicates that protein oxidation enhances substrate recogn ition for the proteasome. In cont rast, the influence of protein oxidation on substrate recognition for calpains or caspase-3 remains unknown and this forms the basis for the current experiments 18

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Figure 2-1. Pathway of myofilament release by calpain followed by actin and myosin degradation by the proteasome. Note that increases in intracellular Ca2 + concentration activate calpain which degrades myofibrillar protein substrates releasing the myofilaments. 19

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Figure 2-2. Pathways leading to the activati on of caspase-3 during MV: 1) Mitochondrial pathway occurs through the release of cyto chrome c and activation of caspase-9. 2) Calcium releasing pathway results in increases in intracellular Ca2 + concentration activating calpain and caspase-12. 20

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CHAPTER 3 MATERIALS AND METHODS This chapter will be divided into two secti ons. Section one includes the experimental design used in each of our experiments that are intended to determine if oxidative modification of myofibrillar proteins increases their suscepti bility to degradation by calpain and/or caspase-3. In the subsequent section, we will provide the methodological details associated with each experimental protocol an d biochemical technique. Experiment 1: Animals Animal Model Justification To address our specific aim and determine if oxi datively modified myofibrillar proteins are more vulnerable to degradation by caspase-3 and calpain (I and II), adult (4-6 month old) female Sprague-Dawley (SD) rats were used. The animal s were 4-6 months of age (young adult) at the time of sacrifice. The SD rat was chosen due to the similarities be tween the rat and human diaphragm in both anatomical and physio logical parameters (2, 3, 54, 56, 60, 61, 95). Animal Housing and Diet All animals were housed at the University of Florida Animal Care Services Center according to guidelines set forth by the Institutional Animal Care and Use Committee. The Animal Care and Use Committee of the Universi ty of Florida approved these experiments. Animals were maintained on a 12:12 hour reve rse light-dark cycle and provided food (AIN93 diet) and water ad libitum throughout the experimental period. Experimental Design Adult rats were randomly assigned to two pr imary experimental gr oups: 1) control (CON; n=8)), 2) oxidation (H2O2 and Fe2+; n=8). The costal diaphragm was removed from each animal the myofibrillar proteins were isolated for subsequent biochemical assays. Group 1 was divided 21

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further into four additional groups; these groups included a co ntrol group, a group treated with active caspase-3, active cal pain I, and active calpa in II respectively. Grou p 2 contained 3 groups. Each myofibrillar protein sample was exposed to different oxidizing treatments resulting in a low, moderate, and high level of protein oxidation. The three levels of oxidation were chosen in order to simulate the level of protein oxidation observed following 6, 12, and 18 hours of MV. The oxidation levels for the three groups were: 1) Low (25 M H2O2 and 10 M Fe2+) 2) Moderate (25M H2O2 and 25 M Fe2+) 3) High (25M H2O2 and 50 M Fe2+). Each of these three groups was then independent ly treated with eith er active caspase-3, active calpain I, or active calpain II (Figure 3-1). Each group had a sample size n=8 chosen based upon a power analysis from preliminary data. Animal Protocol Animals in each group were acutely anesthetized with sodium pentobarbital (60 mg/kg IP). After reaching a surgical plane of anesthesia the animals were sacrif iced and the diaphragm was immediately frozen in liqui d nitrogen and stored at -80 C for subsequent analyses. Statistical Analysis Comparisons between groups were made by a one-way ANOVA and when appropriate simple main effects tests and Tukey HSD tests we re performed. Significance was established at P < 0.05. General Methods Biochemical Measurements Myofibrillar isolation. Samples were prepared based on the method of Reid et al. 1994 (70). Diaphragm samples were first homogeni zed in a buffer containing 0.039 M sodium borate (pH 7.1), 0.025 M KCl, 5mM ethelyne glycol-bis( -aminoethyl ether)-N,N,N N-tetraacetic acid (EGTA) and a protease inhibitor cocktail (Sig ma). The homogenate was then centrifuged at 4C 22

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for 12 minutes at 1500g. After centr ifugation, the supernatant was discarded and the pellet was resuspended and homogenized again. The second homogenization buffer contained: 100mM KCl and 1.0% Triton X-100. This process was repeated twice. After the final centrifugation the final pellet was obtained and resuspended in 0.4 M KCl, 50 mM tris(hydroxymethyl)-aminomethane (Tris) (pH 7.4) and 1.0 mM dithiothreitol (DTT). Protein concen tration was then determined (Bradford). Reactive carbonyl derivatives. Reactive carbonyl derivative s were assessed using the Oxyblot Oxidized Protein Det ection Kit from Chemicon International (Temecula, Ca) as described by the manufacturer. Myofibrillar protein samples were treated with one of four differing levels (Control, Low, Mode rate and High) of the oxidants H2O2 and Fe2+ according to the experimental design and incubated at 37C for 15 minutes. Samples were then immediately cooled to 4C in order for the oxidation to be terminated. Samples were separated via polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and incubated with primary antibody in order to confirm four distinct levels of oxidation. Peptide mapping via gel electrophoresis. Peptide mapping was used to investigate the fragmentation pattern generated by digestion of myofibrillar proteins by each protease. Briefly, 20 l of each of the myofibrillar protein sa mples was treated independently with 2 l of either active calpain I, calpain II, or caspase-3 and 2 l of 50 M Ca2+. Samples were then incubated at 37C for 30 minutes. Samples were then immediately cooled to 4C in order to terminate the reaction. Samples were separated via polyacrylamide gel electrophoresis and then stained with Coomassie Blue. The gels were then analyzed using Image J software in order to determine percentage change from control. The protein bands chosen for analysis were at the molecular weights of 250, 100, 37 and 30 kDa. 23

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Western blot analysis. Proteins were separated via pol yacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, nonspecific sites were blocked for 2 h at room temperature in a PBS solution containing 0.05% Tween-20 and 5% non-fat milk. Membranes were then incubated overnight at 4C with primary antibodies di rected against the protein of interest. The myofibrillar prot eins myosin, actin, troponin I, and -actinin were all probed as a measurement of specific protein degradation. Myosin heavy chain was probed using 1:1000 dilution of a monoclonal antibody obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa Department of Biological Sciences. Actin wa s incubated with a 1:400 dilution of primary polyclonal antibody (Santa Cruz Biotechnology ). Troponin I was incubated with a 1:1000 dilution of polyclonal antibody (S anta Cruz Biotechnology), and -actinin was incubated with a 1:500 dilution of primary polycl onal antibody (Santa Cruz Biotechnology). Following incubation membranes were washed extensively with PBS-Tween and either sheep anti-mouse (myosin) or donkey anti-rabbit (actin, troponin I and -actinin) IgG horseradish peroxidase (amersham biosciences) diluted 1:2000. After washing, a ch emiluminescent system was used to detect labeled proteins (GE healthcare ) and membranes were developed using autoradiography film and a developer (Kodak). 24

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Isolated Myofibrillar Proteins Control n=8 Low n=8 High n=8 Moderate n=8 Active Calpain I Active Caspase-3 No Treatment Active Calpain II Active Calpain I Active Caspase-3 Active Calpain II Active Calpain I Active Caspase-3 Active Calpain II No Oxidation Oxidation Control n=8 Active Calpain I Active Caspase-3 Active Calpain II Biochemical assays Oxidative Stress -Peptide Mapping Reactive Carbonyl Derivatives Myofibrillar Substrates Figure 3-1. Experimental design fo r investigating the effects of various levels of myofibrillar protein oxidation on protein degrad ation by caspase-3 and calpains. 25

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CHAPTER 4 RESULTS Redox Balance Our experimental plan required the production of different levels of oxidized myofibrillar proteins. To achieve this goal, we exposed isolated myofibrillar proteins to three different concentrations of H2O2 and Fe2+. Protein carbonyl formation (i.e reactive carbonyl derivatives; RCD) is an excellent biomarker of protein oxidation and was used in our experiments to document the level oxidative damage to myofibrillar proteins. Oxyblot. Our results revealed that our lowest level of protein oxidation resulted in a significant increase in RCD formation when comp ared with control (P<0.001) (Figure 4-1). Moreover, the moderate oxidation treatment produced slightly higher leve ls of RCD formation when compared to both control and low oxi dation (P<0.001and P<0.01), respectively. Finally, our high oxidation treatment resulted in further elevated levels of RCDs when compared to control, low and moderate oxidation (P<0.001, P< 0.001, P<0.05), thus showing distinct levels of oxidation. Myofibril Digestion Peptide mapping provides a method to quantif y the controlled cleav age of myofibrillar proteins by individual proteases. Therefore, pe ptide mapping was used to identify protein degradation based upon a comparison between cont rol samples (no exposure to proteases) and myofibrillar samples that were incubated with individual proteases. Polyacrylamide SDS-page gels were used in order to obtain molecular weight information about the peptides produced (Figure 4-2). By visual inspection, we identified numerous protein bands that were degraded by both calpain (I and II) and capase-3. From these bands, we selected four prominent bands for 26

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quantitative analysis. A summary of the impact of myofibrillar protein oxidation on proteolytic degradation in these selected myofibrillar proteins follows. 250-kDa protein band. Compared to control, the 250 kD a molecular weight protein was decreased in band size and intens ity when independently exposed to calpain I, calpain II, and caspase-3 (Figure 4-3). Indeed, all three proteases signifi cantly degraded the 250 kDa band. Importantly, as the protein oxidation level in creased, the degradati on of the 250 kDa band increased in all proteases. Speci fically, compared to control, i ndependent exposure of the highest oxidized proteins to calpain I, ca lpain II, and caspase-3 resulted in degradation of more than 90% of the 250 kDa band. 100-kDa protein band. Protease degradation of a 100 kDa protein band was also significantly affected by increased oxidative modi fication (Figure 4-4). In the protein samples treated with caspase-3, each treatment resulted in significant in creases in protein degradation. Both the moderate and high oxidation groups were significantly different from the no oxidation group. Caspase-3 degradation of the 100-kDa band ranged from 19% (no oxidation treatment) to 73% degradation (high oxidation treatment). The protein samples exposed to calpain I ranged in degradation from 23% to 71% as the level of oxidation increased fr om no oxidation to high oxidation. Similarly, exposure of protein samples to calpain II resulted in significant protein degradation ranging from 12% to 73% (i.e., low oxidation to high oxidation). 37-kDa protein band. The protein band at a molecular weight of ~37-40 kDa was degraded by all three proteases in a similar manner to the 250 and 100 kDa bands (Figure 4-5). Specifically, increased protein oxidation increa sed protein breakdown by calpain I, calpain II, and caspase-3. 27

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30-kDa protein band. At the molecular weight of ~30 kDa there was a pair of protein bands that were substrates for both calpain and caspase-3 (Figures 4-2 an d 4-6). Similar to the three previous proteins, increase d oxidation resulted in a genera l increase in breakdown of these ~30 kDa proteins by calpains and caspase-3. Identification of Specific Protein Substrates To identify the specific proteins that calpain and caspase-3 were degrading we employed a Western blotting technique with antibodies dire cted toward four known substrates of calpain and caspase-3. These blots were then analyzed to determine the impact of protein oxidation on proteolytic degradation by calpain and caspase-3. Myosin heavy chain. Myosin heavy chain was identifie d via monoclonal antibodies as the dominant protein band located at ~250 kDa. Myosin was degraded by both calpains and caspase-3 and oxidation increased the magnitude of degradation (Fi gure 4-7). Specifically, control (non-oxidized) m yosin was 29% depleted with the addition of active caspase-3 and this protein continued to be degraded to a greater extent as the protei n oxidation level increased (i.e., 85% degradation with high oxidation). Similar to the caspase-3 mediated degradation of myosin, both calpain I and calpain II degrad ation of myosin was also increas ed as a function of the level of protein oxidation. Actin. Actin was identified as the major protein located at ~37-40kDa. Similar to myosin, actin was degraded by all three proteases and the magnitude of degrad ation increased as a function of the level of prot ein oxidation (Figure 4-8). -actinin. -actinin was recognized as the promin ent protein band located at ~100 kDa. This protein was degraded by bot h calpains and caspase-3, and oxi dation increased the level of degradation via all three prot eases. More specifically, each level of protein oxidation significantly increased th e susceptibility of -actinin to be degraded by caspase-3. In contrast, 28

PAGE 29

moderate-to-high levels of oxi dation were required to increase the susceptibility of -actinin to degradation by calpain I and II (Figure 4-9). Troponin I. The ~25 kDa protein band was identified as troponin I. Troponin I showed great susceptibility to degradation when exposed to both calpain I and II. Moreover, oxidation increased the vulnerability of this protein to calpain-mediated proteolysis (Figure 4-10). Troponin I was also a substrate for caspase-3 an d the level of degradation was accelerated by oxidation. Note, however, that compared to the calpains, the magnitude of caspase-3-mediated troponin I degradation was significantly less. 29

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A B Figure 4-1. Assessment of the le vel of reactive carbonyl deriva tives (RCD) in myofibrillar proteins exposed to varying levels of oxidizing treatments. A) Western blot to determine the level of RCD in myofibrillar protein isolated from the rat diaphragm. The control sample (left) was not exposed to oxidants. The samples in lanes 2, 3, 4 were exposed to three levels of H2O2 and Fe2 + (i.e. 1) low, 2) moderate, 3) high). These results clearly indicate that the oxidi zing treatment resulted in increased levels of protein oxidation in myof ibrillar protein. Low = 25m H2O2 and 10 m Fe2 +, moderate = 25m H2O2 and 25m Fe2 +, and high =25m H2O2 and 50 m Fe2 +. B) Fold difference (versus control) of the oxi dation levels. *low oxidation significantly increased versus control (p<0.001). #modera te oxidation signif icantly increased versus control and low oxidation (p< 0.001, p<0.01). high oxidation significantly increased from control, low and mo derate oxidation (P<0.001, P<0.001, P<0.05). 30

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Figure 4-2. Isolated myofilaments from diaphragm mu scle were either not oxidized or exposed to three levels of oxidation (l ow, moderate, high) via H2O2 and Fe2 + prior to exposure to caspase-3 and calpain (I and II). Samples were then separated via SDS-PAGE and stained with Coomassie Blue. The figure depicts an increase in degradation of specific proteins with an increa se in the level of oxidation. 31

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A B C Figure 4-3. 250 kDa band intensity (percent diffe rence versus control) Values are mean percentage change SE. A) *Low oxid ation significantly increased versus no oxidation (P<0.001). Moderate oxidation signi ficantly increased versus no oxidation (P<0.001). High oxidation significantly increa sed from no oxidation and low oxidation (P<0.001, P<0.01). B) *Low signifi cantly increased versus no oxidation (P<0.05). Moderate significantly incr eased versus no oxidation (P<0.001). High significantly increased from no oxidati on and low oxidation (P<0.001, P<0.05). C) Moderate significantly increase d versus no oxidation (P<0.001). High significantly increased from no oxidation a nd low oxidation (P<0.001, P<0.01). 32

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A B C Figure 4-4. 100 kDa band intensity (percent diffe rence versus control) Values are mean percentage change SE. A) Moderate oxidation significantly increased versus no oxidation (P<0.001). High oxidation significantly increased from no oxidation, low oxidation and moderate oxidation (P<0.001, P<0.001, P<0.05). B) #Moderate significantly increased versus no oxi dation and low oxidation (P<0.01, P<0.05). High significantly increased from no oxidation and low oxidation (P<0.001, P<0.001). C) #Moderate significantly incr eased versus no oxidation and low oxidation (P<0.001, P<0.01). High significantly increased from no oxidation low oxidation and moderate oxi dation (P<0.001, P<0.001, P<0.05). 33

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A B C Figure 4-5. 37 kDa band intensity (percent difference versus control). Values are mean percentage change SE. A) *Low oxid ation significantly increased versus no oxidation (P<0.05). #Moderate oxidation signif icantly increased versus no oxidation and low oxidation (P<0.001, P<0.05). High oxidation significantly increased from no oxidation and low oxidation (P<0.001, P<0.001). B) #Moderate significantly increased versus no oxidation and low oxidation (P<0.01, P<0.05). High significantly increased from no oxidati on and low oxidation (P<0.001, P<0.001). C) Moderate significantly increase d versus no oxidation (P<0.01). High significantly increased from no oxidation a nd low oxidation (P<0.001, P<0.01). 34

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A B C Figure 4-6. 30 kDa band intensity (percent difference versus control). Values are mean percentage change SE. A) *Low oxidati on significantly increased from no oxidation (P<0.05). Moderate oxidation significantly increased versus no oxidation (P<0.001). High significantly increased from n o, low and moderate oxidation (P<0.001, P<0.001, P<0.01). B) *Low significantly in creased from no oxidation (P<0.01). Moderate significantly increased versus no oxidation (P<0.001). High significantly increased from no, low and moderate oxidation (P<0.001, P<0.001, P<0.05). C) Moderate significantly increase d versus no oxidation (P<0.001). High significantly increased from no and low oxidation (P<0.001, P<0.001). 35

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A B C Figure 4-7. Myosin protein (percen t difference versus control). Values are mean percentage change SE. A) *Low oxidation significantly increased from no oxidation (P<0.01). #Moderate significantly increased vers us no and low oxidation (P<0.001, P<0.05). High significantly increased from n o, low and moderate oxidation (P<0.001, P<0.001, P<0.05). B) *Low significantly in creased from no oxidation (P<0.01). #Moderate significantly increased vers us no and low oxidation (P<0.001, P<0.05). High significantly increased from n o, low and moderate oxidation (P<0.001, P<0.001, P<0.05). C) *Low significantly in creased from no oxidation (P<0.001). Moderate significantly increase d versus no oxidation (P<0.001). High significantly increased from no and low oxidation (P<0.001, P<0.001). 36

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A B C Figure 4-8. Actin protein (percent difference versus control). Values are mean percentage change SE. A) *Low oxidation significantly increased from no oxidation (P<0.05). Moderate oxidation significantly in creased versus no oxidation (P<0.001). High oxidation significantly increased from no and low oxidation (P<0.001, P<0.05). B) Moderate significantly increased versus no oxidation (P<0.01). High significantly increased from no oxidation (P<0.001). C) M oderate significantly increased versus no oxidation (P<0.05). High significantly increased from no oxidation and low oxidation (P<0.001, P<0.05). 37

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A B C Figure 4-9. -actinin protein (percent difference versus control). Values are mean percentage change SE. A) *Low oxidation significantly increased from no oxidation (P<0.001). Moderate oxidation significantly in creased versus no oxidation (P<0.01). High oxidation significantly increased from no oxidation and low oxidation (P<0.001, P<0.05). B) Moderate significantly incr eased versus no oxidation (P<0.01). High significantly increased from no oxida tion and low oxidation (P<0.001, P<0.001, P<0.05). C) High significantly increased from no oxidation (P<0.01). 38

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A B C Figure 4-10. Troponin protein (per cent difference versus control) .Values are mean percentage change SE. A) Moderate significantly increased versus no oxidation (P<0.05). High significantly increased from no oxi dation (P<0.01). B) Moderate significantly increased versus no oxidation (P<0.01). High significantly increased from no oxidation (P<0.001). C) Mode rate significantly increa sed versus no oxidation (P<0.05). High significantly increa sed from no oxidation (P<0.01). 39

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CHAPTER 5 DISCUSSION Overview of Principal Findings These experiments provide new and important information regarding the effects of myofibrillar protein oxidation on the susceptibility to degradation by the proteases calpain (I and II) and caspase-3. We tested the hypothesis th at protein oxidation-i nduced modification of myofibrillar proteins would increase their suscepti bility to degradation by calpain and caspase-3. Our findings support this postulate as increa sed levels of protein oxidation augmented myofibrillar protein cleavage and degradation. A detailed discussi on providing an interpretation of our experiments follows. Calpains and Caspase-3 Release Myofilaments The ubiquitin-proteasome system has been shown to be a major proteolytic system activated in skeletal muscle dur ing periods of disuse. Evidence to support this statement comes from two key observations: 1) atrophying muscles contain an accumulation of ubiquitin conjugated proteins and increased proteasome activity; and 2) pharmacological inhibition of proteasome activity retards disuse muscle at rophy (5, 43, 65, 79, 86). However, the proteasome is incapable of breaking down in tact actomyosin complexes, whic h constitute the bulk of muscle protein (42, 55, 78). Therefore, di sassociation of actomyosin complexes appears to be the ratelimiting step in muscle pr otein degradation (78). Growing evidence indicates that both calpain (I and II) and caspase-3 are capable of degrading intact actomyosin complexes. In this regard, Mitch and colleagues were the first to report that caspase-3 is capabl e of cleaving actomyosin complexes and myosin (14). These results suggest that caspase-3 may play a criti cal role in the initiation of muscle protein degradation because release of actomyosin complexes is required for subsequent protein 40

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degradation by the ubiquitin proteasome system (55). Moreover, evidence also indicates that increased calpain activity promotes degradation of many structural and regulatory myofibrillar proteins (8, 20). This is significan t because calpain activity is elev ated in skeletal muscles during disuse and calpain activation lowe rs the levels of the cytoskelet al protein desmin in muscles exposed to prolonged peri ods of unloading (15). To examine whether calpain (I and II) and caspase-3 are capable of cleaving intact actomyosin we performed preliminary experiments by treating intact actomyosin with active forms of each protease, and measuring protei n degradation via peptide mapping. Our findings revealed that intact actomyosin is degraded by both calpains and caspase-3 (data not shown). Induction of Oxidative Stress during Disuse Atrophy Previous work from our lab has revealed that MV promotes diaphragmatic oxidative injury (7, 10, 16, 52, 53, 64, 74, 76, 91, 94). Therefore we postulated that oxi dative modification of myofibrillar proteins during MV and other models of disuse atrophy caused these proteins to be more readily recognized and degraded by calpain (I and II) and caspase-3. To test this postulate, we oxidized myofibrillar proteins to va rying degrees to replicate the three different levels of oxidative damage observed following 6, 12, and 18 hours of MV. Our results confirm that our experimental protocol was successful in oxidizing myofibrillar proteins to achieve three significantly different levels and that as protein oxidation increased, so did protein degradation by both calpain and caspase-3. This important finding will be discussed in detail in the next segment. Oxidation of Myofibrils Increases Protein Degradation by Calpain and Caspase-3 Muscle proteins are differentially susceptible to oxidative modi fication during exposure to oxidants (27, 94). Our lab has previously s hown that prolonged MV promotes oxidation of numerous diaphragmatic proteins including actin and myosin (94). Both actin and myosin are 41

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degraded by calpains at a slow rate, however when these substrates become chemically modified they become more rapidly degrad ed by calpain I and II (19-22). Our peptide maps of the proteolytic digest s supported our hypothesis that oxidative modification of myofibrillar proteins increases the susceptibi lity of numerous proteins to degradation by calpain (I and II) and caspase-3. Compared to control, oxidized myofibrils exhibited an enhanced rate of protein br eakdown at many molecula r weights (e.g., 250, 100, 37, 30 kDa). In general, the magnitude of protein degradation increased as a function of the level of oxidation. To identify the specific proteins that were undergoing degradation, we used monoclonal antibodies against four different proteins that ma tched the molecular weights of the protein bands that were degraded by calpains and caspase-3. Th is analysis revealed that the 250 kDa band was myosin heavy chain, the 100 kDa band was -actinin, and the 37 kDa band was actin. Troponin I was also probed because it is a known substrate of each protease. Our results show that increased oxidative modification to each of these proteins resulted in increased degradation by both calpain (I and II) and caspase-3. Although some variatio n existed among these proteins, all of these substrates showed that increasing levels of oxidation from low to high resulted in a progressive rise in protein degradation. Conclusions and Future Directions In summary, our data demonstrate that ca lpain (I and II) and caspase-3 rapidly degrade numerous myofibrillar proteins. It also reveals that the pr otein oxidation resulting from prolonged periods of disuse is capable of enhanc ing the vulnerability of numerous proteins to degradation by calpain and caspase -3. These results also highlight the importance in attenuating oxidative stress during skeletal mu scle disuse in order to decrease the enhanced cleavage of sarcomeric proteins and subsequently decr ease the rate of di suse-induced atrophy. 42

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43 Although it is now clear that oxidative stress promotes disu se muscle atrophy, numerous unresolved issues exist. For example, are disuse -induced redox disturbanc es responsible for the activation of both calpain and caspase-3 in skel etal muscles? Moreover, future studies should address the possibility that cross-talk may exist between calpain and caspase-3. That is, theoretically, active caspase-3 can promote cal pain activation and vice-versa. Therefore, determining if there is potential for synergistic activity between these two proteases would assist in developing a genetic or pharm acological approach to inhibiting one or both of these proteases during muscle wasting conditions. Another import ant area for future studies would be to determine the site of oxidant production in skeletal muscles during prol onged disuse. Identifying the pathway(s) responsible for oxidant production in inactive skeletal muscle would provide the insight needed for the development of optimal therapeutic strategies to prevent or retard disuseinduced oxidant damage in skeletal muscle. Indeed, improving our understanding of the cell signaling pathways that regulat e disuse muscle atrophy remains an exciting area for future research.

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13. Deruisseau KC, Shanely RA, Akunuri N, MT, Van Gammeren D, Zergeroglu AM, McKenzie M, and Powers SK. Diaphragm unloading via contro lled mechanical ventilation alters the gene expression profile. Am J Respir Crit Care Med 172: 1267-1275, 2005. 14. Du J, Wang X, Miereles C, Bailey JL, Debi gare R, Zheng B, Price SR, and Mitch WE. Activation of caspase-3 is an initial step trigge ring accelerated muscle proteolysis in catabolic conditions. J Clin Invest 113: 115, 2004. 15. Enns DL, Raastad T, Ugelstad I, Belcastro AN. Calpain/calpastatin activities and substrate depletion patterns during hindlimb unw eighting and reweighting in skeletal muscle. Eur J Appl Physiol 2007 Jul;100(4):445-55. 16. Falk DJ, Deruisseau KC, Van Gammere n DL, Deering MA, Kavazis AN, and Powers SK. Mechanical ventilation promotes redox status alterations in the diaphragm. J Appl Physiol 101: 1017-1024, 2006. 17. Furuno K and Goldberg AL. The activation of protein de gradation in muscle by Ca2+ or muscle injury does not involve a lysosomal mechanism. Biochem J 237: 859-864, 1986. 18. Goll DE, Dayton WR, Singh I, and Robson RM. Studies of the alpha-actinin/actin interaction in the Z-disk by using calpain. J Biol Chem 266:8501-8510, 1991. 19. Goll DE, Neti G, Mares SW, and Thompson VF. Myofibrillar prot ein turnover: the proteasome and the calpains. J Anim Sci, 2007. 20. Goll DE, Thompson VF, Li H, Wei W, and Cong J. The calpain system. Physiol Rev 83: 731-801, 2003. 21. Grune T and Davies KJ. The proteasomal system and HNE-modified proteins. Mol Aspects Med 24: 195, 2003. 22. Grune T, Merker K, Sandig G, and Davies KJ. Selective degrada tion of oxidatively modified protein substrates by the proteasome. Biochem Biophys Res Commun 305:709-718, 2003. 23. Hasselgren PO and Fischer JE. The ubiquitin-proteasome pathway: review of a novel intracellular mechanism of musc le protein breakdown during sepsis and other catabolic conditions. Ann Surg 225: 307, 1997. 24. Hasselgren PO and Fischer JE. Muscle cachexia: current concepts of intracellular mechanisms and molecular regulation. Ann Surg 233: 9-17, 2001. 25. Hasselgren PO, Wray C, and Mammen J. Molecular regulation of muscle cachexia: it may be more than the proteasome. Biochem Biophys Res Commun 290: 1-10, 2002. 45

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51. McClung JM, Kavazis AN, DeRuisseau KC, Falk DJ, Deering MA, Lee Y, Sugiura T, and Powers SK. Caspase-3 regulation of diaphragm myonuclear domain during mechanical ventilation-induced atrophy. Am J Respir Crit Care Med 175: 150-159, 2007. 52. McClung JM, Kavasis AN, Whidden MA, De Ruisseau KC, Falk DJ, Criswell DS, and Powers SK. Antioxidant administration attenuates mechanical ventilation-induced rat diaphragm muscle atrophy independent of protein kinase B (PKB Akt) signaling. J Physiol 585: 203-215, 2007) 53. McClung JM, Whidden MA, Kavasis AN, Falk DJ, Deruisseau KC, and Powers SK. Redox regulation of diaphragm proteoly sis during mechan ical ventilation. Am J Phsiol Regul Integr Comp Physiol 2008. 54. Metzger JM, Scheidt KB, and Fitts RH. Histochemical and physiological characteristics of the rat diaphragm. J Appl Physiol 58: 1085-1091, 1985. 55. Mitch WE and Goldberg AL. Mechanisms of muscle wasting: The role of the ubiquitinproteasome pathway. N Engl J Med 335: 1897-1905, 1996. 56. Mizuno M. Human respiratory muscles: fi bre morphology and capillary supply. Eur Respir J 4: 587-601, 1991. 57. Munoz KA, Satarug S, Tischler ME. Time course of the res ponse of myofibrillar and sarcomeric protein metabolism to unweighting of the soleus muscle. Metabolism 42: 1006-1012, 1993. 58. Nader GA, Hornberger TA, and Esser KA. Translational control: implications fro skeletal muscle hypertrophy. Clin Orthop Relat Res : S178-187, 2002. 59. Nader GA, McLoughlin TJ, and Esser KA. mTOR function in skeletal muscle hypertrophy: increased ribosomal RNA via cell cycle regulators. Am J Physiol Cell Physiol 289: C1457-1465, 2005. 60. Poole DC, Sexton WL, Farkas GA, Powers SK, and Reid MB. Diaphragm structure and function in health and disease. Med Sci Sports Exerc 29: 738-754, 1997. 61. Powers SK, Demirel HA, Coombes JS, Fl etcher L, Calliaud C, Vrabas I, and Prezant D. Myosin phenotype and bioenergetic charac teristics of rat respiratory muscles. Med Sci Sports Exerc 29: 1573-1579, 1997. 62. Powers SK, Kavazis AN, and DeRuisseau KC. Mechanisms of disuse muscle atrophy: role of oxidative stress. Am J Physiol Regul Integr Comp Physiol 288: R337-344, 2005. 63. Powers SK, Kavazis AN, and McClung JM. Oxidative stress and disuse muscle atrophy. J Appl Physiol 2007. 48

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64. Powers SK, Shanely RA, Coombes JS, Koesterer TJ, McKenzie M, Van Gammeren D, Cicale M, and Dodd SL. Mechanical ventilation result s in progressive contractile dysfunction in the diaphragm. J Appl Physiol 92: 1851-1858, 2002. 65. Price SR, Bailey JL, Wang X, Jurkovitz C, England BK, Ding X, Phillips LS, Mitch WE. Muscle wasting in insulinope nic rats results from activ ation of the ATP-dependent, ubiquitin-proteasome proteolytic pathway by a mechanism including gene transcription. J Clin Invest 98(8):1703-8, 1996. 66. Price SR, Du JD, Bailey JL, and Mitch WE. Molecular mechanisms regulating protein turnover in muscle. Am J Kidney Dis 37: S112-114, 2001. 67. Primeau AJ, Adhihetty PJ, and Hood DA. Apoptosis in heart and skeletal muscle. Can J Applied Physiol 27: 349-395, 2002. 68. Purintrapiban J, Wang M, and Forsberg NE. Degradation of sarcomeric and cytoskeletal proteins in cu ltured skeletal muscle cells. Comp Biochem Physiol B Biochem Mol Biol 136: 393-401, 2003. 69. Rando TA. Oxidative stress and the pathoge nesis of muscular dystrophies. Am J Phys Med Rehabil 81: S175-186, 2002. 70. Reid MB. Response of the ubiquitin-proteasome pa thway to changes in muscle activity. Am J Physiol Regul Integr Comp Physiol 288: R1423R1431, 2005. 71. Reid MB, Andrade FH, Balke CW, and Esser KA. Redox mechanisms of muscle dysfunction in inflammatory disease. Phys Med Rehabil Clin N Am 16: 925-949, ix, 2005. 72. Reid WD, Huang J, Bryson S, Walker DC, and Belcastro AN. Diaphragm injury and myofibrillar structure induced by resistive loading. J Appl Physiol 76:176-184. 73. Rivett AJ. Preferential degradati on of the oxidatively modified forms of glutamine synthetase by intracellular mammalian proteases. J Biol Chem 260: 300-305, 1985. 74. Shanely RA, Coombes JS, Zergerogl u AM, Webb AI, and Powers SK. Short-duration mechanical ventilation enhances diaphragmatic fatigue resistance but impairs force production. Chest 123: 195-201, 2003. 75. Shanely RA, Van Gammeren D, Deruisse au KC, Zergeroglu AM, McKenzie MJ, Yarasheski KE, and Powers SK. Mechanical ventilation depresse s protein synthesis in the rat diaphragm. Am J Respir Crit Care Med 170: 994-999, 2004. 76. Shanely RA, Zergeroglu MA, Lennon SL, Sugiura T, Yimlamai T, Enns D, Belcastro A, and Powers SK. Mechanical ventilation-induc ed diaphragmatic atrophy is associated with oxidative injury and increased proteolytic activity. Am J Respir Crit Care Med 166: 1369-1374, 2002. 49

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90. Trappe TA, Burd NA, Louis ES, Lee GA, and Trappe SW. Influence of concurrent exercise or nutrition countermeasures on thigh and calf muscle size and function during 60 days of bed rest in women. Acta Physiol (Oxf) 191: 147-159, 2007. 91. Van Gammeran D, Falk DJ, Deering MA Deruisseau KC, and Powers SK. Diaphragmatic nitric oxide synthase is not induced during mechanical ventilation. J Appl Physiol 102: 157-162, 2007. 92. Ventadour S and Attaix D. Mechanisms of skeletal muscle atrophy. Curr Opin Rheumatol 18: 631-635, 2006. 93. Wray CJ, Sun X, Gang GI, and Hasselgren PO. Dantrolene downregulates the gene expression and activity of the ubiquitin-proteasome proteolytic pathway in septic skeletal muscle. J Surg Res 104: 82, 2002. 94. Zergeroglu MA, McKenzie MJ, Shanely RA, Van Gammeren D, DeRuisseau KC, and Powers SK. Mechanical ventilation-induced oxidative stress in the diaphragm. J Appl Physiol 95: 1116-1124, 2003. 95. Zhang P, Chen X, and Fan M. Signaling mechanisms involved in disuse muscle atrophy. Med Hypotheses 2007. 96. Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S, Lecker SH, Goldberg AL. FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metab 6(6): 472-83, 2007. 51

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52 BIOGRAPHICAL SKETCH Ashley Joslin Smuder was born in Naples, Fl orida. She earned a Bachelor of Science degree in exercise physiology from the University of Florida. After graduation, she pursued a masters degree in exercise physiology and began he r graduate work at the University of Florida in 2007 under the direction of Scott K. Powers. Ashley focused her studies on oxidative stress and proteolysis of the diaphragm during prolonged mechanical ventilation. She received her Master of Science degree in 2008.