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Mechanisms of Disuse Muscle Atrophy

Permanent Link: http://ufdc.ufl.edu/UFE0045243/00001

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Title: Mechanisms of Disuse Muscle Atrophy
Physical Description: 1 online resource (118 p.)
Language: english
Creator: Talbert, Erin E
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: atrophy -- casting -- disuse -- muscle -- ros -- ventilation
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Health and Human Performance thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Decreases in skeletal muscle size can occur in response to both disease and muscle inactivity. Two common causes of inactivity-induced muscle atrophy are limb immobilization due to casting and mechanical ventilation (MV), which results in diaphragm inactivity.  Both of these types of muscle atrophy impair affected patient’s quality of life, and limb muscle atrophy can increase the risk of falls. At present, the mechanisms leading to disuse atrophy are poorly understood and therefore, no pharmacological treatment is currently available.Therefore, the purpose of these experiments was three-fold: 1) to determine the role that immobilization-induced increases in mitochondrial reactive oxygen species (ROS) emission play  in the activation of major proteolytic systems, and in the down-regulation of anabolic signaling in locomotor skeletal muscles; 2) to establish the contribution of calpain and caspase-3  in immobilization-induced limb muscle atrophy; and 3) to determine if calcium leak through the ryanodine receptor (RyR) of the sarcoplasmic reticulum is responsible for the increased mitochondrial ROS production, calpain activation,and diaphragm atrophy that occurs during prolonged MV. Our results reveal that immobilization-induced increases in mitochondrial ROS emission are required for activation of all four key proteolytic systems in immobilized limb muscle andthe depression of anabolic signaling that occurs during hindlimb immobilization. Further, both active calpain and caspase-3 are required for disuse atrophy in response to hindlimb casting. Finally, pharmacological blockade of the RyR is not sufficient to prevent MV-induced increases in mitochondrial ROS emission or MV-induced diaphragm atrophy, but is sufficient to prevent the mitochondrial dysfunction resulting from prolonged MV. Collectively, these experiments expand the current understanding of the mechanisms by which disuse atrophy occurs.
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 Erin E Talbert.
Thesis: Thesis (Ph.D.)--University of Florida, 2013.
Local: Adviser: Powers, Scotty K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045243:00001

Permanent Link: http://ufdc.ufl.edu/UFE0045243/00001

Material Information

Title: Mechanisms of Disuse Muscle Atrophy
Physical Description: 1 online resource (118 p.)
Language: english
Creator: Talbert, Erin E
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: atrophy -- casting -- disuse -- muscle -- ros -- ventilation
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Health and Human Performance thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Decreases in skeletal muscle size can occur in response to both disease and muscle inactivity. Two common causes of inactivity-induced muscle atrophy are limb immobilization due to casting and mechanical ventilation (MV), which results in diaphragm inactivity.  Both of these types of muscle atrophy impair affected patient’s quality of life, and limb muscle atrophy can increase the risk of falls. At present, the mechanisms leading to disuse atrophy are poorly understood and therefore, no pharmacological treatment is currently available.Therefore, the purpose of these experiments was three-fold: 1) to determine the role that immobilization-induced increases in mitochondrial reactive oxygen species (ROS) emission play  in the activation of major proteolytic systems, and in the down-regulation of anabolic signaling in locomotor skeletal muscles; 2) to establish the contribution of calpain and caspase-3  in immobilization-induced limb muscle atrophy; and 3) to determine if calcium leak through the ryanodine receptor (RyR) of the sarcoplasmic reticulum is responsible for the increased mitochondrial ROS production, calpain activation,and diaphragm atrophy that occurs during prolonged MV. Our results reveal that immobilization-induced increases in mitochondrial ROS emission are required for activation of all four key proteolytic systems in immobilized limb muscle andthe depression of anabolic signaling that occurs during hindlimb immobilization. Further, both active calpain and caspase-3 are required for disuse atrophy in response to hindlimb casting. Finally, pharmacological blockade of the RyR is not sufficient to prevent MV-induced increases in mitochondrial ROS emission or MV-induced diaphragm atrophy, but is sufficient to prevent the mitochondrial dysfunction resulting from prolonged MV. Collectively, these experiments expand the current understanding of the mechanisms by which disuse atrophy occurs.
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 Erin E Talbert.
Thesis: Thesis (Ph.D.)--University of Florida, 2013.
Local: Adviser: Powers, Scotty K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045243:00001


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1 MECHANISMS OF DISUSE MUSCLE ATROPHY By ERIN ELIZABETH TALBERT 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 2013

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2 2013 Erin Elizabeth Talbert

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3 To Mom and Dad for alway s believing I could do anything

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4 ACKNOWLEDGMENTS First, I would like to thank my mentor, Dr. Scott Powers, for everything. I would also like to thank the members of my committee, Dr. Stephen Dodd, Dr. David Criswell, Dr. Sally Johnson, and Dr. Andrew Judge for their time, guidance, and support. Additionally, I would like to thank Dr. Tom Clanton for his guidance, s upport, and help with the experimental design and procedures for this project and I would like to thank Dr. Peter Adhihetty for his help interpreting the results of these experiments I greatly appreciate the help and support of the Powers lab members and the rest of the APK graduate students Additionally, I would like to thank Kim Hatch, Karen Kleis, and the rest of the staff of APK. Finally, I would like to thank my family and friends for all of their love and support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATION S ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 14 2 LITERATURE REVIEW ................................ ................................ .......................... 18 Limb Immobilization as a Cause of Disuse Atrophy ................................ ................ 18 Mechanical Ventilation Promotes Diaphragmatic Atrophy ................................ ...... 20 Mechanisms Responsible for Disuse Muscle Atrophy ................................ ............ 22 Proteolytic Systems in Disuse ................................ ................................ .......... 23 Cell Signaling Pathways Involved in the Activation of Muscle Proteolytic Systems ................................ ................................ ................................ ........ 27 Oxidative stress in disuse atrophy ................................ ............................. 27 Mitochondrial ROS production ................................ ................................ ... 28 Calcium as a Cause of Increased Mitochondrial ROS Production .................... 29 Summary ................................ ................................ ................................ ................ 30 3 IMMOBILIZATION IND UCED ACTIVATION OF K EY PROTEOLYTIC SYSTEMS IN SKELETAL MUSCLES IS PREVENTED BY A MITOCONDRIA TARGETED ANTIOXIDANT ................................ ................................ ................... 31 Methods ................................ ................................ ................................ .................. 32 Animals and Experimental Design ................................ ................................ .... 32 Tissue Harvesting ................................ ................................ ............................. 33 Biochemica l Measures ................................ ................................ ..................... 34 Results ................................ ................................ ................................ .................... 37 SS 31 Treatment of Ambulatory Control Animals Does Not Alter Muscle Fiber Size or Mitochondrial ROS Emission ................................ ................... 37 SS 31 Attenuates Casting induced Increases in Mitochondrial ROS Emission ................................ ................................ ................................ ........ 37 SS 31 Prevents Casting induced Atrophy ................................ ........................ 37 SS 31 Treatment Prevents the Mitochondrial Dysfunction Induced by Casting ................................ ................................ ................................ .......... 38 SS 31 Prevents Casting induced Calpain Activation ................................ ........ 38

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6 SS 31 Prevents Casting induced Caspase 3 Activation ................................ ... 39 Activation of the Proteasome System Requires Mitochondrial ROS Production ................................ ................................ ................................ ..... 39 Mitochondrial ROS Stimulates Autophagy During Hindlimb Casting ................ 40 SS 31 Does Not Protect Muscle Size by Promoting PGC ........ 41 SS 31 Treatment Protects Markers of Anabolic Signaling During Casting ....... 41 Discussion ................................ ................................ ................................ .............. 42 Conclusions ................................ ................................ ................................ ...... 44 4 CALPAIN AND CAPASE 3 PLAY REQUIRED ROLE S IN IMMOBILIZATION INDUCED LIMB MUSCLE ATROPHY ................................ ................................ .... 55 Methods ................................ ................................ ................................ .................. 57 Animals and Experimental Design ................................ ................................ .... 57 Pharmacological Inhibitors ................................ ................................ ............... 58 Ti ssue Harvesting ................................ ................................ ............................. 59 Biochemical Measures ................................ ................................ ..................... 59 Results ................................ ................................ ................................ .................... 62 Independent Inhibition of Calpain and Caspase 3 Prevents Casting induced Muscle Atrophy ................................ ................................ ............................. 62 Casting Activates Calpain in the Soleus during 7 days of Immobilization ......... 63 Casting Activates Caspase 3 in the Soleus during 7 days of Immobilization .... 63 Potential Mechanism(s) Responsible for Regulat ory Cross talk between Calpain and Caspase 3 ................................ ................................ ................. 64 Inhibition of both Calpain and Caspase 3 Protect against Immobilization induced Mitochondrial Dysfunction ................................ ................................ 65 Discussion ................................ ................................ ................................ .............. 65 Overview of Major Findings ................................ ................................ .............. 65 Independent Inhibition of Calpain or Caspase 3 Activity Prev ents Immobilization induced Soleus Muscle Atrophy ................................ ............ 66 Regulatory Cross talk Exists between Calpain and Caspase 3 ........................ 68 Protection of Mitochondrial Function by Protease Inhibition ............................. 69 Conclusions ................................ ................................ ................................ ...... 70 5 BLOCKAGE OF THE RYAN ODINE RECEPTOR DURIN G MECHAN ICAL VENTILATION IS NOT S UFFICIENT TO PREVENT ATROPHY OR INCREASED MITOCHONDR IAL REACTIVE OXYGEN SPECIES PRODUCTION ................................ ................................ ................................ ........ 77 Methods ................................ ................................ ................................ .................. 79 Animals ................................ ................................ ................................ ............. 79 Experimental Protocol ................................ ................................ ...................... 80 Details of MV ................................ ................................ ................................ .... 80 Tissue Harvesting ................................ ................................ ............................. 81 Functional Measurements ................................ ................................ ................ 81 Mitochondrial Measurements ................................ ................................ ........... 81 Hi stological Measurements ................................ ................................ .............. 83

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7 Biochemical Measurements ................................ ................................ ............. 83 Electron Microscopy ................................ ................................ ......................... 83 Statistical Analysis ................................ ................................ ............................ 84 Results ................................ ................................ ................................ .................... 84 Biological Response to MV ................................ ................................ ............... 84 Impact of MV and AZ on Contractile Function ................................ .................. 84 ROS Production from Permeabilized Diaphragm Fibers ................................ .. 85 MV induced Proteolytic Activity ................................ ................................ ........ 85 MV induced Diaphragm Muscle Atrophy ................................ .......................... 85 Mitochondrial Function ................................ ................................ ..................... 86 Autophagy ................................ ................................ ................................ ........ 86 SR Structural Changes following Prolonged MV ................................ .............. 87 Discussion ................................ ................................ ................................ .............. 87 MV induced ROS Emission, Protease Activation, and Atrophy in the Diaphragm ................................ ................................ ................................ ..... 88 Prevention of MV induced Mitochondrial Dysfunction by Azumolene ............... 88 MV induced SR swelling in Diaphragm fibers ................................ ................... 89 Experimental Limitations ................................ ................................ .................. 90 Conc lusions ................................ ................................ ................................ ...... 91 6 DISCUSSION ................................ ................................ ................................ ....... 102 LIST OF REFERENCES ................................ ................................ ............................. 105 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 118

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8 LIST OF TABLES Table page 3 1 Mitochondrial function of permeabilized soleus fiber bundles. ............................... 48 3 2 Mitochondrial function of permeabilized plantaris fiber bundles. ............................ 48 4 1 Mitochondrial function. ................................ ................................ ........................... 76 5 1 Body weights. ................................ ................................ ................................ ......... 93 5 2 Blood gas measurements during MV. ................................ ................................ .... 93 5 3 Mitochondrial function. ................................ ................................ ........................... 99

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9 LIST OF FIGURES Figure pag e 3 1 Experimental Design to address Aim 1. ................................ .............................. 45 3 2 R eactive oxygen species emission ................................ ................................ ..... 46 3 3 Cross sectional area and representative images. ................................ ............... 47 3 4 Calpain activation and activity ................................ ................................ ............ 49 3 5 Caspase 3 activation and activity; caspase 9 activation ................................ ..... 50 3 6 E3 Ligase expression ................................ ................................ ......................... 51 3 7 Markers of autophagy ................................ ................................ ......................... 52 3 8 PGC ................................ ................................ ............................ 53 3 9 Markers of anabolic signaling ................................ ................................ ............. 54 4 1 Experimental Design to address Aim 2 ................................ ............................... 71 4 2 Cross sectional area and representative images ................................ ................ 72 4 3 Activation and ac tivity of calpain and caspase 3 ................................ ................ 73 4 4 Expression of Bid, tBid, and the ratio of tBid/Bid ................................ ................ 74 4 5 Expression of Calpastatin and Total Calpain. ................................ ..................... 75 5 1 Experimental Design to address Aim 3. ................................ .............................. 92 5 2 Diaphragm Contractile Function. ................................ ................................ ........ 94 5 3 Soleus Contractile Function ................................ ................................ ................ 95 5 4 Reactive oxygen species emission. ................................ ................................ ... 96 5 5 Activity of calpain and caspase 3 ................................ ................................ ...... 97 5 6 Cross sectional area and representative images. ................................ ............... 98 5 7 Representative EM pictures of A) Veh B) MV C) AZ D) MVA ........................... 100 5 8 Ratio of LC3 II/I ................................ ................................ ................................ 101 5 9 Expression of calsequestrin ................................ ................................ .............. 101

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10 LIST OF ABBREVIATION S ADP Adenosine diphosphate ANOVA Analysis of v ariance ATP A denosine triphosphate AZ Azumolene cDNA Complementary deoxyribose nucleic acid CSA Cross sectional area ETC Electron transport chain FoxO Forkhead Box O ( FoxO ) HRP Horseradish peroxidase LC3 Microtubule associated protein light chain 3 MAFbx Muscle Atrophy F box mRNA Messenger ribonucleic acid MuRF 1 Muscle Ring Finger protein 1 MV Mechanical ventilation NADPH N icotinamide adenine dinucleotide phosphate NF kB Nuclear factor kappaB OCT Optimal cutting temperature medium PaCO2 Partial pressure of carbon dioxide PaO2 Partial pressure of oxygen PCR Polymerase chain reaction PE Phosphatidylethanolamine PGC Peroxisome proliferator activated receptor gamma coactivator 1 alpha RCR Respiratory control ratio RNA Ribonucleic acid

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11 ROS Reactive oxygen species RT PCR Reverse transcriptase polymerase chain reaction RyR Ryanodine receptor VEH Vehicle treated VIDD Venti lator induced diaphragm dysfunction

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12 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 DISUSE MUSCLE ATRO PHY By Erin Elizabeth Talbert May 2013 Chair: Scott K. Powers Major: Health and Human Performance Decreases in skeletal muscle size can occur in response to both disease and muscle inactivity. Two common causes of inactivity induced muscle atrophy are limb immobilization due to casting and mechanical ventilation (MV), which results in diaphragm inactiv quality of life, and limb muscle atrophy can increase the risk of falls. At present, the mechanisms leading to disuse atrophy are poorly understood and therefore, no pharmacological trea tment is currently available. Therefore, the purpose of these experiments was three fold: 1) to determine the role that immobilization induced increases in mitochondrial reactive oxygen species (ROS) emission play in the activation of major proteolytic sy stems, and in the down regulation of anabolic signaling in locomotor skeletal muscles; 2) to establish the contribution of calpain and caspase 3 in immobilization induced limb muscle atrophy; and 3) to determine if calcium leak through the ryanodine recep tor (RyR) of the sarcoplasmic reticulum is responsible for the increased mitochondrial ROS production, calpain activation, and diaphragm atrophy that occurs during prolonged MV. Our results reveal that immobilization induced increases in mitochondrial ROS emission are required for activation of all four key

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13 proteolytic systems in immobilized limb muscle and the depression of anabolic signaling that occurs during hindlimb immobilization. Further, both active calpain and caspase 3 are required for disuse atro phy in response to hindlimb casting. Finally, pharmacological blockade of the RyR is not sufficient to prevent MV induced increases in mitochondrial ROS emission or MV induced diaphragm atrophy, but is sufficient to prevent the mitochondrial dysfunction re sulting from prolonged MV. Collectively, these experiments expand the current understanding of the mechanisms by which disuse atrophy occurs.

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14 CHAPTER 1 INTRODUCTION Skeletal muscle atrophy results in muscle weakness and can occur in response to a variety o f diseases (i.e., diabetes and cancer) and muscle in activity. Atrophy induced by reduced muscle contractile activity and decreased muscle loading is referred muscle (93, 95) Although people of all ages are susceptibl e to disuse atrophy, muscle weakness in the elderly is an important health care problem, as older individuals often struggle to recover from periods of decreased muscle activity (24) Frailty of the elderly often results from the combination of disuse muscle atrophy combined with age related muscle loss (i.e., sarcopenia) (56) Understanding the mechanisms by which disuse atrophy occurs will allow the development of countermeasures to preserve muscle mass and quality of life in affected individuals (24) A frequent cause of disuse muscle atrophy in humans is limb immobilization due to racture to heal. One to six weeks of leg cas ting, either in healthy people or following a tibia fracture, significantly decreases muscle fiber size and muscle strength (1, 14, 23, 127) In general, the magnitude of muscle atrophy increases as a function of the du ration of i mmobilization and depends upon the fiber composition of the inactive muscle (23) Mechanical ventilation (MV) is a clinical tool used to maintain adequate alveolar ventilation in pa tients that are incapable of maintaining blood gas homeostasis on their own. Common indications for MV include surgery and patients experiencing acute respiratory failure (44) While M V can be a life saving intervention, prolonged MV promotes the rapid development of atrophy and contractile dysfunction in the primary

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15 muscle of inspiration, the diaphragm (52, 55, 68, 89) This unique and clinically relevant type of disuse atrophy, termed ventilator induced diaphragm dysfunction (VIDD) is associated with prolonged stays in the intensive care unit resulting in large healthcare costs each year (141) Understanding the mechanisms by which disuse atrophy proceeds is an important part o f designing effective countermeasures to prevent this muscle weakness. Mechanistic studies of human at rophy are difficult to complete due to ethical concerns. Many animal models of disuse have been developed, including rodent models of both limb casting an d MV, to allow for both invasive procedures and the use of pharmacological inhibitors not approved for human use (93, 95) The Sprague Dawley rat is often the animal skeletal muscles contain similar structure and fiber type composition as human skeletal muscle (78, 82, 91) Although our understanding of the cellular events leading to disuse muscle atrophy is growing a detailed understanding of the mechanisms responsibl e for disuse muscle atrophy is not currently available Increases in proteolytic activity appear to play an important role in disuse muscle atrophy (124) Further, emerging evidence indicates that reactive oxygen species (ROS), and particularly ROS produced by the mitochondria, are required signals for the development of disuse atrophy induced by both prolonged MV and hindlimb casting (10, 60, 7 9, 92, 133) The experiments contained within this dissertation tested three different hypotheses related to th e signaling pathways by which disuse atrophy proceeds, using both a rat hindlimb casting model and a rat model of MV. The overarching goal of these

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16 experiments was to better understand the mechanism(s) by which disuse atrophy proceeds. Specific Aim 1: To d etermine if treatment with a mitochondria targeted antioxidant during prolonged hindlimb casting of rats is sufficient to maintain anabolic signaling and prevent activation of all four major proteolytic systems in skeletal muscle during prolonged inactivit y Rationale: Previous work in our laboratory has demonstrated that treatment of mice with the mitochondria targeted antioxidant SS 31 is sufficient to prevent casting induced muscle atrophy in mice (79) However, it remains unknown if a mitochondrial targeted ant ioxidant can protect rat skeletal muscle against disuse muscle atrophy. Hypotheses: Autophagy, the calpains, caspase 3, and the ubiquitin proteasome system of proteolysis are all activated by limb casting, and treatment with SS 31 will attenuate the activ ation of each of these proteolytic systems. Additionally, treatment with SS 31 will prevent the casting induced decreases in anabolic signaling in the affected skeletal muscles Specific Aim 2: To determine if inhibit ion of the proteases calpain or caspase 3 is sufficient to prevent atrophy induced by prolonged hindlimb casting. Rationale: Pharmacological inhibition of calpain or caspase 3 has been shown to be sufficient to prevent the diaphragmatic atrophy and contractile dysfunction resulting from 12 hou rs of MV (85) However, t he role that these proteases play in hindlimb disuse atrophy remains unknown

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17 Hypothesis: Calpain and caspase 3 play important roles in the skeletal muscle atrophy induced by hindlimb casting, and inhibition of these proteases will be sufficient to prevent or impede this atrophy. Specific Aim 3: To establish the role that calcium release from the sarcoplasmic reticulum (SR) plays in diaphragm mitochondrial ROS emission, fiber atrophy, and contractile dysfunction during prolonged MV. Rationale: The SR and mitochondria are tethered together in muscle cells such that calcium release from the SR i s sufficient to increase metabolic flux through the Krebs cycle, raising the mitochondrial membrane potential and consequently increasing mitochondrial ROS production. While it is established that prolonged MV results in increased mitochondrial ROS emissio n in diaphragm muscle fibers, the mechanism(s) responsible for this increase is unclear. Hypothesis: Calcium release from the SR via the ryanodine receptor (RyR) contributes to increased diaphragm mitochondrial ROS emission during MV, leading to MV i nduced diaphragm atrophy and contractile dysfunction.

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18 CHAPTER 2 LITERATURE REVIEW Skeletal muscle atrophy is defined as a decrease in the size of individual fibers in a muscle. Many conditions, including disease, malnutrition, and muscle inactivity cause s keletal muscle atrophy. Muscle inactivity, also termed muscle disuse, includes atrophy that results from conditions such as spaceflight, where prolonged weightlessness decreases load bearing by the joints and muscles, and denervation, where muscles are no longer controlled by nerves (93, 95) Disuse m uscle atrophy includes situations that making it likely that some muscle activity does occur (39, 42) An example of reduced use is a associated with a broken bone A lthough the leg is in a cast to prevent movement and allow healing, the individual can still wiggle their toes which involves activity of some muscles. The purpose of this review is to provide a brief overview of topics ge rmane to the current understanding of disuse muscle atrophy. Subjects include discussions of both limb immobilization and mechanical ventilation, which are two causes of disuse atrophy. Additional areas addressed include a summary of the cellular mechanism s leading to disuse atrophy, including the pr oteolytic systems responsible for degrading muscle protein and the sources of oxidative stress which are responsible for activating these proteases. Finally, the potential impact of increased cytosolic calcium on mitochondrial function and reactive oxygen species emission will be detailed. Limb Immobilization as a Cause of Disuse Atrophy Muscle weakness has long been known to be a consequence of prolonged immobilization (i.e., casting) of a limb. Several studies have quantified the effects of

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19 casting, determining both the amount of atrophy in several different muscles and the effects on the contractile pr operties of these muscles. As little as one week of leg cast immobilization in patients with a fractured tibia is sufficient to decrease the cross sectional area of Type I muscle fibers of the vastus lateralis muscle, with six weeks of casting inducing an approximately 30% decrease in the size of both Type I and Type II muscle fibers (14) Other studies using healthy volunteers have found similar results, with atrophy incre asing in response to longer periods of immobilization [reviewed in (1) ]. Additionally, this disuse induced decrease in muscle fiber size is also accompanied by a decrease in the force produced by a maximal voluntar y contraction [ reviewed in (1) ]. This combination of decreased muscle size and force production results in substantial muscle weakness, and can be a contributing factor to frailty, particularly in the elderly (56) Interestingly, older adults appear to be more susceptible to immobilization induced atrophy than younger adults (127) underscoring the importance of developing effective countermeasures to prevent disuse atrophy. Because ethica l and practical considerations make studying disuse atrophy in humans difficult, several animal models have been developed to study this problem, including rodent models of casting (93, 95) Animal models also have the advantage of allowing researchers to use interventions to prevent atrophy that could not ethically be used in humans, such as gene overexpression or pharmaceutical compounds not approved for human use. Rodents have similar skeletal muscle characteristics to humans, and the mechanism of atrophy appears to be similar (54)

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20 Hindlimb casting of rodents results in significant atrophy in both the so leus and plantaris after 7 days of immobilization when the ankle is fixed in a plantar flexed position (28, 29, 79, 100, 111, 112) Significant contractile dysfunction also occurs in rodent muscles that have undergone prolonged immobilization (120) These decreases in specific force production likely result from both changes within the muscle and is extended (40) It should be noted that disagreement exists in the literature about if casting induced models of atrophy should involve the casting o f either one or both limbs. Proponents of a model where only one leg is casted argue that the contra lateral (non casted) limb provides the best possible control because this limb is exposed to the same metabolic, cytokine, and hormonal milieu as the exper imental (casted) limb (71) Proponents of a model using two casted limbs argue that the contra lateral limb can experience compensatory hypertrophy due to increased usage of the uncasted limb (7) This is a significant potential problem that questions th e wisdom of using a one leg casting model to investigate disuse muscle atrophy. Mechanical Ventilation Promotes Diaphragmatic Atrophy Mechanical ventilation (MV) is a life saving intervention to maintain adequate alveolar ventilation in patients who canno t do so on their own. Many circumstances can lead to patients requiring ventilator assistance to maintain blood gas homeostasis, including anesthetics used during surgery, respiratory failure, heart failure, and damage to the nervous system that leaves the patient unable to breathe on their own (126) In performing the re quired level of alveolar ventilation to maintain adequate pulmonary gas

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21 exchange. In these cases, MV becomes a life saving intervention. During full support MV, the completely inactive and the ventilator is doing all of th e work of breathing (51) This decrease in resp iratory muscle activity results in inactivity induced inspiratory muscle atrophy (52, 55, 68) The diaphragm is the primary mus cle of inspiration, does most of the work of breathing in humans and atrophies rapidly during prolonged MV (93 95, 97) It is important to note that ventilator support to patients can be classified into two general categories: 1) full ventilator support (control MV) ; or 2) partial ventilator support. When a patient is ventilated using full respiratory support the ventilator provides all of during partial ventilator support, the ventilator provides a portion of the work of breathing respiratory muscles (i.e., diaphragm) provide the remainder. Partial support MV is a more common clinical mode of MV world wide (32) but control MV is used when patients are incapable of doing any of the work of breathing. Importantly, both partial support MV and control MV lead to diaphragm atrophy and contractile dysfunction (51, 107, 108) The first suggestio n that human diaphragm atrophy occurs during prolonged MV came from a retrospective study of nec ropsy samples from post mortem infants. In this work, Knisely et al. reported a significant decrease in the cross sectional areas of diaphragm fibers from child ren ventilated greater than 12 days prior to death compared to children ventilated less than 7 days (62) More recently, Levine et al. reported a ~50% decrease in the cross sectional area of both Type I and Type II fibers when comparing the diaphragms of brain dead organ donors who had undergone 18 69 hours of MV to

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22 diaphragms from patients under going short term thoracic surgeries (68) The authors predicted that this atrophy would result in a significant decrease in diaphragm for c e production. Another study by Jaber et al. confirmed the decrease in muscle fiber size following prolonged mechanical ventilation and also found a progressive decrease in diaphragm contractile function during prolonged MV (55) The concept that the magnitude of VIDD increases as a function MV duration is also supported by a study demonstrating a strong posi tive correlation between the maximal diaphragm force production and the total duration of MV (48) This study was conducted on intact diaphragms in human patients and provides a strong argument tha t the level of VIDD is duration dependent. Because of ethical concerns, performing mechanistic studies of VIDD in humans is nearly impossible. Ther efore, to investigate the mechanisms responsible for VIDD, several animal models of MV have been developed (6, 10, 25, 27, 34, 51, 60, 72, 74, 76, 77, 85, 92, 105 109, 113, 114, 132, 133, 140) Importantly, these animal models closely mimic the MV induced diaphragmatic atrophy, contractile dysfunction, and protease activation observed in the human diaphragm during prolonged MV (52, 55, 68, 89) These animal models have proven to be important tools for understanding the mechanisms by which prolonged MV induces diaphragm weakness. In particular, these experimental models permit pharmacological and genetic manipulations to the diaphragm that are not possible in humans. Mechanisms Responsible for Disuse Muscle Atrophy Muscle fiber size is determined by a balance of both protein synthesis and protein degradation. When fiber size remains constant, the rates of protein synthesis and protein degradation are balanced. During muscle fiber atrophy, prote in degradation

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23 increases and protein synthesis decreases (124) Both of these changes are well documented in rat diaphragm muscle fibers during prolonged MV (51, 72, 74, 85, 113) and in rodent limb muscles during prolonged inactivity (e.g., hindlimb casting) (16, 61, 79, 125) Controversy exists about the relative contributions of protein synthesis and protein degradation to the loss of muscle mass in human stud ies (73) but in animal models, the increase in protein degradation appears to play a larger role in muscle atrophy than the decrease in protein synthesis (124) Proteolytic Systems in Disuse Four major proteolytic systems are present in skeletal muscle, and all four appear to be active in skeletal muscles during disuse induced fiber atrophy. A brief discussion of these systems follows. Ubiquitin proteosome system. The ubiquitin proteasome syst em is a widely studied pathway of protein degradation during disuse muscle atrophy. Briefly, the ubiquitin proteasome system consists of 3 enzymes (denoted E1, E2, and E3) which ed proteins are then trafficked to the proteasome, where they are broken into small peptides (80) parts, and is more formally called the 26S pro teasome, which reflects its size. The 26S proteasome includes a 20S proteasome core and two 19S regulatory subunits located on either side of the 20S core (122) The E3 enzymes, and in particular the muscle specific E3 ligases atrogin 1 (also known as Muscle Atrophy F box or MAFbx) and Muscle Ring Finger protein 1 (MuRF 1), have been suggested as the rate lim iting step in the atrophic process (47, 80) Both the Forkhead Box O ( FoxO ) and nuclear factor kappa B ( NF kB ) signaling pathways are

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24 suggested to control the increase in the proteasome acti vity during prolonged disuse (58) Animals with the g enetic knock outs of both atrogin 1 and MuRF 1 are protected against disuse muscle atrophy (15) demonstrating the requirement of both proteins fo r muscle atrophy. Additionally, treating casted animals with the proteasome inhibitor MG132 prevented immobilization induced atrophy (21) Calpain. The calpain system consists of several cysteine proteases (most notably calpains 1, 2, and 3 in muscle) that are activated by increased levels of free calcium in the cytosol (43) It has been reported that calpains do not appear to directly degrade actin and myosin (43, 50, 122) II spectrin) which provide structure to myofilaments are known substrates of calpain (50) Additionally, although native actin and myosin are not substrates of calpain, oxidized actin and myosin can both be degraded by calpain (116) Pharmacological inhibition of cal pain has been shown to inhibit MV induced diaphragm atrophy and contractile dysfunction (72, 85) Thus, calpain appear s to be an induced hindlimb atrophy remains less clear. Calpain is known to be activated in hindlimb immobilization (79) and also during hindlimb suspension, another model of disuse atrophy (31) Two st udies using hindlimb suspension overexpressed the endogenous inhibitor of calpain, calpastatin. One study reported significant protection against disuse induced atrophy (125) while the other failed to find significant protection against atrophy, but did obser ve a preservation of muscle specific force (102) Thus, more work remains to be done to determine what role calpains play in hindlimb disuse atrophy.

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25 Caspase 3. Caspase 3 is one of many members of the caspase family of prote ases. Th is cysteine protease is best known for its role in apoptosis, specifically as (69) and has also been demonstrated to play a key role in disuse skeletal muscle atrophy. Pharmacological inhibition of caspase 3 can prevent MV induced diaphragm atrophy and contractile dysfunction (74, 85) Further, c aspase 3 activation has been shown to increase the (30) suggesting that caspase 3 activation may be upstream of the proteasome during atrophy and contractile dysfunction indu ced by prolonged MV. Although pharmacological inhibition studies have demonstrated the importance of both calpain and caspase 3 in skeletal muscle, the exact roles that calpain and caspase 3 play in disuse muscle atrophy remain s debatable. Increased expres sion of the muscle specific E3 ligases (e.g. atrogin 1 and MuRF 1) is the rate limiting step of the proteasome system (65, 119) and many investigators pred ict that the prote asome system is the rate limiting step of muscle protein degradation during disuse muscle atrophy (122) Indeed, the muscle proteins myosin and actin appear to be largely degraded by the proteasome system (122) However, it appears that the ubiquitin proteasome system cannot degrade actin and myosin when these proteins are in intact sarcomeres (30, 119) II spectrin and talin serve to keep actin and myosin appropriately aligned in the myofilaments. Calpain is known to cleave II spectrin and talin (43, 50) myosin f rom the sarcomere, allowing the E3 ligases to ubiquitinate these proteins and

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26 target them for degradation by the proteasome Caspase 3 may act in a similar manner to calpain and also release actin and myosin for subsequent degradation (30) Au tophagy system. degrades and recycles damaged organelles and cytosolic proteins. In healthy cells, autophagy is critical for recycling old or damaged organelles and making room for new organelles (9) Recently, new evidence suggests that the autophagy system plays a role in skeletal muscle protein degradation. Specifically, during times of muscle stress and inactivity autophagy is upregulated and therefore may contribute to protein degradation (9, 103) Autophagy functions by forming a double wall vesicle ( autophagosome ) around organelles or proteins to be degraded (134) The autophagosome then fuses with a lysosome, allowing the lysosomal proteases (i.e. cathepsins) to degrade the contents of the autophagosome (134) Prolonged MV is known to increase the expression of many autophagy genes in human diaphragm (52) and also in a rat model of VIDD (Smuder unpublished data). How autophagy interacts with the other proteolytic systems during disuse atrophy in limb muscle and particularly in VIDD remains unknown. One study using a mouse model concluded that two weeks of casting did not increase the expression of autophagy related genes (13) ; however, this study was conducted late in the atrophic process, so autophagy signaling could have increased early in the atrophy process but returned to baseline le vels prior to the completion of these experiments Clearly, more res earch is required to determine the precise role that autophagy plays in disuse muscle atrophy.

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27 Cell Signaling Pathways Involved in the Activation of Muscle Proteolytic Systems All four proteolytic systems in the diaphragm appear to be activated by the incr ease in reactive oxygen species (ROS) during prolonged MV (60, 92, 133) The likely source of this ROS and its role in activating the proteases required for disuse atrophy is discussed in the next section. Oxidative stress in disuse atrophy Disuse muscle atrophy has long been associated with an increase in bio markers of oxidat ive stress, and increases in ROS production appear to be responsible for the activation of proteolytic signaling during disuse [reviewed in (98) ]. Our laboratory has investigated several potential sources of th is increase in ROS (i.e., Fenton chemistry with free iron, the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme nitric oxide synthase, the enzyme xanthine oxidase, and the mitochondria) in skeletal muscles undergoing disuse at rophy (93) A series of experiments has determined that oxidants resulting from free iron, NADPH oxidase, nitric oxide synthase, and xanthine oxidase do not play major roles in oxidant production in the diaphragm during p rolonged MV (35, 76, 128, 132) In contrast mitochondria appear to be a major source of ROS producti on in the diaphragm during prolonged MV. Indeed, compared to control animals, Kavazis et al. demonstrated an approximately 25% increase in hydrogen peroxide released from mitochondria isolated from the diaphragm of animals exposed to 12 hours of MV (60) This increase in ROS release occurred during both state 3 (ADP stimulated respiration) and stat e 4 respiration (basal state). Additionally, this increase in mitochondrial ROS emission was accompanied by a decrease in mitochondrial coupling, suggesting that damage occurs in diaphragm mitochondria during MV. When animals are treated with a mitochondri a

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28 targeted antioxidant during 12 hours of mechanical ventilation, the atrophy and contractile dysfunction associated with VIDD are prevented (92) Additionally, prevention of MV induced increases in mitochondrial ROS emission prevents the activation of calpain, caspase 3, and the proteasome system in diaphragm muscle fibers (92) Finally, the increase in autophagy signaling in the diaphragm that is normally observed during prolonged MV does not occur when animals ar e treated with a mitochondrial targeted antioxidant (Smuder unpublished data). Similar results have been observed in a mouse model of hindlimb casting (79) Therefore, it i s important to determine the mechanism responsible for MV induced increases in mitochondrial ROS production M itochondrial ROS p roduction As the powerhouse of the cell, mitochondria produce adenosine triphosphate (ATP), which is the stored form of energy used by cells. Mitochondria generate a potential across their membrane and use this potential to phosphorylate adenosine diphosphate (ADP) to ATP. Carriers such as NADH and FADH 2 donate electrons to the electron transport chain (ETC), where a collection of membrane proteins are used to ia are highly efficient at moving ETC. These electrons are often accepted by O 2 to form the superoxide radical (O 2 ). Superoxide is an oxygen radical that is quickly co nverted to the non radical oxidant hydrogen peroxide (H 2 O 2 ) by superoxide dismutase. Once formed, if catalase or glutathione peroxidase do not convert H 2 O 2 to water, H 2 O 2 can diffuse throughout the cell and contribute to redox sensitive cell signaling proc esses. During prolonged periods of disuse, muscles are not contracting and require significantly less ATP than during

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29 activity. ETC leak, and consequently muscle mitochondrial ROS release, is known to increase when ATP demand is low (18) As a result, an increase in mitochondrial ROS production during muscle disuse is predicted. T he mechanism for this increase remains unclear, but r ecent evidence suggests that increases in cytosolic calcium conc entrations may be responsible for stimulating muscle mitochondria to produce more ROS (20) Calcium as a Cause of Increased Mitochondrial ROS Production Throughout skeletal muscle, intracellular calcium is an important signaling molecule, including in the mitochondria. During prolonged periods of inactivity, cytosolic calcium rises in skeletal muscle fibers (53, 59) As cytosolic calcium rises, some of this calcium is driven into the mitochondrial matrix. Amongst other things, increases in mitochondrial Ca 2+ concentrations lead to increased electron flow through the ETC (110) When muscles are contracting this increased ETC flux is beneficial because energy demand is increasing. However, during disuse, energy demand is not increasing. Because no ADP is available to phosphorylate, the energy potential across the mitochondrial membran e continues to rise (110) An increase in mitochondrial membrane potential increases electron leak, leading to increases in mitochondrial ROS production (66, 81) A potential source of the increased cytosolic calcium during prolonged muscle disuse is leakage from the sarcoplasmic reticulum via the ryanodine receptor (RyR). The RyR is a gated channel that allows calcium release from the sarcoplasmic reticulum into the cytosol to promote muscle contraction. Oxidation of the RyRs causes calcium leak in skeletal muscles of senescent animals (4) In this regard, stabilization of RyRs using a pharmacological agent is sufficient to prevent this age induced calcium leak and preserve muscle specific force (4) As discussed previously oxidative stress plays a

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30 major role in disuse atrophy, and oxidation of the RyR may be responsible for the increased intracellular calcium concentrations observed during disuse atrophy. Additionally, as discussed above, increased cytosolic calcium leads to the activation of calpain, which is a critical protease in several models of disuse atrophy. Thus, it is feasible that prevention of the calcium leak through the RyR may be sufficient to prevent disuse atrophy by both protecting mitochondria from calciu m overload and prevention of calpain activation in muscle. Summary Prolonged skeletal muscle inactivity results in muscle fiber atrophy. This type of skeletal muscle atrophy is an important clinical problem, with limb muscle atrophy contributing to frailty and MV induced diaphragm atrophy resulting in diaphragm weakness and increasing the risk for ventilator dependency. The e xperiments described in this dissertation were designed to further delineate the mechanisms contributing to disuse atrophy using both a rodent model of limb ca sting and a rodent model of MV.

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31 CHAPTER 3 IMMOBILIZATION INDUCED ACTIVATION O F KEY PROTEOLYTIC SY STEMS IN SKELETAL MUSCLES IS PREVENTED BY A MITOC ONDRIA TARGETED ANTIOXIDANT Prolonged periods of s keletal muscle inactivity can lead to muscle fiber atrophy Common human conditions leading to d isuse atrophy include limb immobilization and prolonged bedrest. Because m uscle atrophy studies often require invasive tissue sampling, animal models of disuse muscle atrophy have been developed (e.g., h indlimb cast immobilization of rodents used to mimic human limb immobilization ) (95) Currently details of the signaling pathways leading to disuse atrophy remain largely unknown. Understanding these mecha nisms is important, as currently no effective countermeasures exist to combat disuse atrophy (84) Disuse muscle atrophy results from decrease d protein s ynthesis and increase d protein degradation, with degradation accounting for the majority of the atrophy (124) Four key proteolytic systems exist in skeletal muscle: autoph agy, the ubiquitin proteasome system, the calpain system, and the caspase system. All four of these proteolytic systems are active during disuse atrop hy in humans and animals (95) but the relative contribution of each system to disuse atrophy remains unknown O xidative stress play s a major rol e in the disuse atrophy process and activates both calpain and caspase 3 in muscles exposed to prolonged inactivity (79, 92, 133) Mitochondrial oxidant production (e.g., superoxide and resulting hydrogen peroxide) appears to be a required upstream signal to promote calpain and caspase 3 activation in several models of muscle disuse, including hindlimb immobilization of mice (10, 64 79, 133) Currently, little is known about the redox sensitivity of the proteasome and the autophagy system during hi ndlimb disuse, although both systems are activated in

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32 diaphragm muscle in response to prolonged mechanical ventilation, another type of disuse muscle atrophy associated with oxidative stress (10, 92, 133) Therefore, we determined if inactivity induced mitochondrial oxidant production is required to activate both the proteasome system and autophagy during disuse muscle a trophy induced by hindlimb immobilization (i.e., casting). Further, we also determined whether inactivity induced oxidative stress in limb muscle plays a required role in the down regulation of anabolic signaling in the inactive muscles. We hypothesized t hat muscle inactivity induced increases in mitochondrial ROS production in limb muscle fibers plays a required role in the increase in the proteolytic activities of the proteasome and autophagy systems along with a decrease in anabolic signaling in muscle. Cause and effect was determined by treating immobilized animals with a mitochondrial targeted antioxidant to scavenge reactive oxygen species produced in mitochondria within the inactive skeletal muscles. Methods Animals and Experimental Design Adult f em ale 300g Sprague Dawley rats (Charles River, Wilmingto n, MA) were assigned to one of 4 groups: 1) non casted (ambulatory) receiving saline injections (Con, n=10), 2) non cast ed (ambulatory) receiving the mitochondria targeted antioxidant SS 31 (Con SS, n=1 0 ), 3) h indlimbs casted receiving saline (Cast, n=8), and 4 ) hindlimbs casted receiving the mitochondria targeted antioxidant SS 31 (Cast SS, n=8), (Figure 3 1). SS 31 was dissolved in saline and given in a daily subcutaneous injection at a dose of 3 mg/kg Saline animals received an equal volume of saline daily. All experiments were approved by the University of Florida Institutional Animal Care and

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33 Use Committee. Importantly, data from these Con and Cast animals has been previously published (123) Immobilization. Animals were ca st ed under the influence of gaseous isoflurane (2% for induction of anesthesia, 0.5 1.5% to maintain a surgical plane of anesthesia). it (3M Health Care, St. Paul, MN) Animals were casted with plaster of Paris (Gypsona, Reynosa, Tamaul ipas, Mexico) from the last rib down through both legs with the ankle plantar flexed as to induce maximum soleus and ability to chew through the cast. Animals were monitored daily to ensure that animals did not experience swollen or abraded feet and did not damage the cast. Additionally, animals were check ed to make sure they had free access to water and food. Tissue Ha rvesting Following 7 days of casting, animals were anesthetized to a surgical plane of anesthesia using isoflurane and the cast was removed. The soleus and plantaris muscles were removed from both hindlimbs, and then the animals were sacrificed. One soleus and one plantaris were split into t w o pieces. One piece of each muscle was dissected into two pieces and permeabilized to measure ROS emission and mitochondrial function as descri bed below. The other half of each plantaris and soleus was embedded in Optimal Cutting Temperature (OCT) medium (Sakura Finetek USA, Torrance, CA) and frozen in li quid nitrogen cooled isopentane as previously described (74) The soleus and plantaris fr om the opposite leg were snap frozen in liquid nitrogen and stored at 80C for Western blotting and real time RT PCR measurements

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34 Biochemical Measures Preparation of permeabilized muscle fibers. M itochondrial ROS release and mitochondrial oxygen consump tion were determined in permeabilized muscle fibers as previously described (79) Briefly, ~10 mg pieces of muscle were teased apart on ice in cold buffer X (60 mM K MES, 35 mM KCl, 7.23 mM K 2 EGTA, 2.77 mM CaK 2 EGTA, 20 mM imidazole, 0.5 mM DTT, 20 mM taurine, 5.7 m M ATP, 15 mM phosphocreatine, and 6.56 mM MgCl 2 pH 7.1). After dissection, muscle fiber membranes were permeabilized bundles were rotated in buffer Z (110 mM K MES, 35 mM KCl, 1 mM EGTA, 5 mM K 2 HPO 4 3 mM MgCl 2 0.05 mM glutamate, 0.02 mM malate, and 0.5 mg/ml BSA, pH 7.1) three times for five minutes to remove all saponin. Mitochondrial ROS production. Peroxide/Peroxidase Assay Kit (Life Techno logies, Grand Island, NY) was used to determine the level of reactive oxygen species release from permeabilized fiber bundles. Fiber bundles were incubated at 37C in 96 well plates in reaction buffer (10 mM MgCl2, 10 mM KH2PO4, 100 mM KCl, 50 mM MOPS, 1 m BSA, 5 mM succinate, 0.02 units HRP, 2.5 ug Amplex Red, pH 7.0) as previously described (79) After 30 minutes of incubation, the amount of fluorescent resorufin produced was measured using an excitation wavelength of 545 nm and an emission wavele ngth of 590 nm in flurometric multiwell plate reader (SpectraMax, Molecular Devices, Sunnyvale, CA). Mitochondrial Oxygen Consumption. Mitochondrial function was measured by determining the rate of oxygen consumption of permeabilized soleus and plantaris f iber bundles using a respiration chamber (Hansatech Instruments) as previously described

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35 (79) Substrate (5 mM pyruvate and 2 mM malate) was added to 1 mL of buffer Z containing 20 mM creatine and the permeabilized fiber bundle. State 3 respiration was stimulated b y 0.25 mM ADP. After 5 minutes, stimulate state 4 respiration. The respiratory control ratio (RCR) was determined by dividing maximal state 3 oxygen consumption by state 4 oxygen consumption. Myofiber Cross Sectional Area OCT embedded pieces of muscle were sliced at dystrophin and myosin heavy chain isofor ms as described previously (74) Cross sectional ar ea was determined by tracing using Scion Image software (Scion Corp., Frederick MD). A minimum of 100 fibers per muscle were analyzed for each animal. Western Blotting Muscles were placed in 5 mM Tris/5 mM EDTA (pH 7.5) 1:10 zed using a motorized glass on glass system. P rotease inhibitor cocktail was used 1:20 (vol/vol) (Sigma Aldrich). Samples were centrifuged at 4C for 10 minutes at 1,500 g to pellet insoluble protein Supernatant p rotein concentration was determined by the method of Bradford (17) Laemmli buffer (Bio Rad, mercaptoethanol was mixed 1:1 with equal amounts of muscle protein. Proteins were separated on 4 20% Tris HCl gels (Bio Rad). After blockin g in Licor Blocking buffer, membranes were incubated with primary antibody against cleaved (active) calpain 1, cleaved caspase 3, cleaved caspase 9, LC3, II spectrin (Santa Cruz B iotechnology, Santa Cruz, CA). Membranes were either visualized using the Odyssey system and fluorescent secondary antibodies (Licor Biosciences, Lincoln, NE) or developed on photographic film using enhanced

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36 chemiluminesce reagents (GE HeathCare, Buckingha mshire, UK). Films were analyzed using the Kodak Image Station 440 and Kodak 1D software. To control for protein tubulin detected on the same membrane. Real Time Reverse Trans criptase PCR. for mRNA isolation and cDNA synthesis has been previously described (117) Briefly, total mRNA was isolated by homogenizing snap frozen muscle tissue in TRIzol reagent (Life Technologies, Carlsbad, CA) according to the manufacturer's directions. Spectrophotometry was used to determine RNA content, and 3 ug of mRNA was reverse transcribed to cDNA using the Superscript II I First Strand Synthesis System (Life Technologies). Taqman chemistry (Applied Biosystems, Foster City, CA) was used to determine the relative expression of our genes of interest using the computed tomography method (Applied Biosystems, User Bulletin no. 2, ABI PRISM 7700 Sequence Detection System) and the ABI Prism 7000 Sequence Detection system (Applied Biosystems) Twenty determine t he expression of atrogin 1 (Entrez Gene ID 171043), MuRF 1 (140939), PGC glucuronidase ( 244434) Our laboratory has previously used glucuronidase as our reference gene based on work showing unchanged expression in mechanical ventilation, which is another type of disuse atrophy (26, 27) Statistical Analysis. Comparisons between groups were made by a one way analysis of variance and, when ap ence test

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37 was performed post hoc. Significance was established at p < .05. Data are presented as means standard error of t he mean. Results SS 31 Treatment of Ambulatory Control Animals Does Not Alter Muscle Fiber Size or Mitochondrial ROS Emission Treatment of ambulatory control animals with SS 31 for seven days did not alter mitochondrial ROS emission from either soleus (Fi gure 3 2A) or plantaris (Figure 3 2B) muscle fiber bundles. Additionally, SS 31 treatment did not alter muscle cross sectional area (CSA) of any fiber type in either the soleus (Figure 3 3A) or the plantaris muscles (Figure 3 3B). Thus, comparisons have be en made only between CON, CAST, and CAST SS groups. SS 31 Attenuates Casting induced Increases in Mitochondrial ROS Emission To assess the effectiveness of SS 31 in prevention of mitochondrial ROS emission, we measured hydrogen peroxide release from permea bilized soleus and plantaris fibers bundles Compared with ambulatory control animals, immobilization resulted in a significant increase in mitochondrial ROS release from both the soleus and plantaris muscles. Treatment with SS 31 attenuated this inactivit y induced increase in mitochondrial ROS emission in the soleus (Figure 3 2A), and completely prevented the increased ROS emission in the plantaris (Figure 3 2B). SS 31 Prevents Casting induced A trophy Seven days of casting induced a significant decrease in the CSA of type I fibers of the soleus, and SS 31 treatment during casting completely prevented this decrease (Figure 3 3 A). No significant differences existed between groups in the CSA of type IIA muscle fibers in the soleus.

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38 In the plantaris muscle, c as ting signi ficantly decreased the size of t ype IIA, type IIB/IIX and hybrid IIA/IIB/IIX fibers (Figure 3 3B ) SS 31 treatment prevented the casting induced decrease in each of these fiber types. No significant differences were observed in the CSA of type I fibers in the plantaris SS 31 Treatment Prevents the Mitochondrial Dysfunction Induced by C asting Prolonged casting of the hindlimb muscles decreased the mitochondrial respiratory control ratio (RCR) in both the soleus and the plantaris muscles (Tables 1 and 2). RCR is the ratio of state 3 ( ADP s timulated) oxygen consumption divided by State 4 ( oligomycin inhibited) oxygen consumption, and decreases in RCR are indicative of impaired mitochondrial coupling Daily SS 31 treatment attenuated the casting indu ced decrease in RCR in both the soleus and plantaris muscle. Additionally, casting induced a significant increase in State 4 respiration in the soleus, which was completely prevented by SS 31 treatment. Finally, State 3 respiration in both plantaris and so leus muscle fibers was higher in CAST SS animals compared to CAST animals. We have previously demonstrated that treating ambulatory animals with SS 31 has no effect on skeletal muscle mitochondrial function (79) SS 31 P r events Casting induced Calpain A ctivation Our laboratory has previously reported that SS 31 prevents the casting induced increase s in calpain activation in both the plantaris and soleus muscles in the mouse (79) We confirm these findings in a rat model of hindlimb muscle disuse (Figures 3 4A and 3 4 B) and additionally demonstrate that SS 31 prevents immobilization induced II spectrin degradation in the soleus and plantaris. When II spectrin, a specific fragment is detectable at 145 k D via Western blot. The presence of this fragment is an index of in vivo calpain

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39 activity over the last several hours of the experiment (129) Casting induced a significant increase in the calpain II spectrin in both the soleus and plantaris; this increase was completely prevented by SS 31 treatment (Figures 3 4 C and 3D). SS 31 Prevents Casting induced Caspase 3 A ctivation Our group has also previously reported that SS 31 prevents the casting induced activation of caspase 3 in a mouse model (79) We confirm this finding in a rat model of muscle atrophy, with SS 31 completely p reventing the increase in active caspase 3 in the soleus (Figure 3 5 A), and attenuating the casting induced increase in active casp ase 3 in the plantaris (Figure 3 5 B). Additionally, we report that the SS 31 treatment attenuated the increase in the caspase 3 specific (120 kD) cleavage fragment II spectrin in the soleus (Figure 3 5 C). Similar to the 145 kD fragment specific to calpain, the presence of this breakdown product is an index of caspase 3 activity over the last several hours of the experiment (129) We were unable to detect significant I I spe ctrin in the plantaris (Figure 3 5 D). Finally, we report that while we found no significant dif ferences in the soleus (Figure 3 5 E), caspase 9 activation increased due to ca sting in the plantaris (Figure 3 5 F), and SS 31 treatment attenuated this incr ease. T his data suggests that caspase 9 may play a role in caspase 3 activation during casting. Activation of the P roteasome S ystem Requires M itochondrial ROS P roduction Seven days of hindlimb casting resulted in a significant increase in the mRNA expres si on of both atrogin 1 (Figure 3 6A) and MuRF 1(Figure 3 6 B) in the soleus, and atrogin 1 (Figure 3 6 C) in the plantaris. In the soleus, these increases in E3 ligase expression were completely prevented by SS 31 treatment. Additionally, the increase in atrog in 1 expression in the plantaris was partially attenuated by daily SS 31 treatment,

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40 while no differences in MuRF 1 expression existed between groups in t he plantaris (Figure 3 6 D). Increased expression of atrogin 1 and MuRF 1 are associated with increased signaling through FoxO3a and NF (58) N o differences in the ratio of phosphorylated FoxO3a to total FoxO3a existed between groups in the soleus or plantaris muscles (data not shown). Additionally, we observed no differences in the of the NF between experimental groups in the soleus or plantaris muscles (data not shown). Mitochondrial ROS Stimulates Autophagy During Hindlimb C asting The lysosomal protein microtubule associated protein light chain 3 (LC3) is conjugated with phosphatidylethanolamine (PE) during autophagy (83) LC3 without PE is called LC3 I, while lipidated LC3 is referred to as LC3 II. An increa se in the ratio of LC3 II/LC3 I is representative of an increased presence of autophagic vesicles, a required part of autophagy. Casting significantly increased the ratio of LC3 II/ LC 3 I in both the soleus (Figure 3 7A) and the plantaris (Figure 3 7 B). Dai ly treatment of animals with SS 31 prevented this increase in LC3 II/LC3 I ratio in both muscles, demonstrating that mitochondrial ROS promotes an increase in the number of autophagic vesicles during casting. An increase in autophagy during casting is supp orted by an increase in the mRNA expression of cathepsin L, a protease involved late in autophagy, in both the soleus and plantaris (Figures 3 7C and 3 7 D). Mitochondrial ROS is required for the increase in cathepsin L, as SS 31 treatment prevents the cast ing induced increase in this protease in both the soleus and plantaris. While not definitive proof of increased autophagy during casting, these data support either increased autophagy or a failure of autophagy to function properly during casting.

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41 SS 31 Doe s Not Protect Muscle Size by Promoting PGC xpression Recent evidence reveals that Ppar gamma co decreased following denervation, and that overexpression of PGC protect muscle against denervation and fasting induced atrophy (104) We hypothesized that mitochondrial ROS may be responsible for the decrease in PGC ed by casting. However, SS 31 treatment did not prevent the decrease in PGC the s oleus or the plantaris (Figure 3 8A and 3 8 B), suggesting that, at least after 7 days of casting, the decrease in PGC SS 31 T reatment Protects M arkers of Anabolic Signaling During C asting Although increase d protein degradation is the predominant mechanism responsible for muscle fiber atrophy during prolonged disuse decreases in muscle protein synthesis also occur, and c ontribute to muscle wasting during prolonged disuse. The proteins Akt and mTOR both play important signaling roles in promoting protein synthesis by regulation of translation Both proteins are activated by phosphorylation at specific sites (Akt at Serine 473, mTOR at Serine 2448), and the ratio of phosphorylated protein to total protein is an index of signaling through these molecules (16) The ratio of pAkt to Akt was decreased by casting in both the soleus and the plantaris, and SS 31 treatment partially attenuated the decrease in pAkt/Akt in both muscles (Figures 3 9A and 3 9 B). In the soleus, the ratio of pmTOR/mTOR decreased in response to 7 days of casting, and this decrease was completely prevented by SS 3 1 treatment during casting (Figure 3 9 C). In the plantaris muscle casting did not decrease pmTOR/mTOR ratio, but treatment with SS 31 during casting significantly increased the ratio of pmTOR/mTOR c ompared to control animals (Figure 3 9 D). Finally, no differences

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42 existed between experiment al groups in the protein levels of pAkt, Akt, pmTOR, or mTOR in either the plantaris or the soleus muscles (data not shown). Discussion These experiments confirm the important role that increased mitochondrial ROS play in promoting disuse muscle atrophy a nd demonstrate the ability of a mitochondrial targeted antioxidant (SS 31) to prevent disuse atrophy of hindlimb muscles in the rat. Importantly, these are the first experiments to demonstrate that increased mitochondrial ROS emission plays a required role in inactivity induced increases in key proteins involved in both the autophagy and the proteasome proteolytic systems in locomotor skeletal muscles A discussion of these and other important findings follows. It is well established that disuse muscle atr ophy is associated with oxidative stress in the inactive skeletal muscles (64) Moreover, recent work has demonstrated that preventing inactivity induced oxidative stress in muscle is sufficient to prevent disuse atrophy (10, 28, 133) Although ROS can be produced at several locations in skeletal muscles (93, 9 5) recent evidence indicates that the m itochondria are in the primary site for ROS in skeletal muscle during prolonged inactivity (79, 92) In the current study, we confirmed that treatment of animals with a mitochondrial targeted antioxidant (SS 31) is sufficient prevent immobilization induced atrophy of the soleus and plantaris. Similar to previous results (79) we also observed that inactivity induced activation of both calpain 1 and caspase 3 in the soleus and plantaris muscles require increased mitochondrial ROS emission. Indeed, treatment of animals with the mitochondrial targeted antioxidant SS 31 prevent s the casting induced activity of both proteases in the soleus muscle, and prevented calpain activation in the plantaris

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43 muscle. This is significant becaus e previous work indicates that the inhibition of calpain or caspase 3 is sufficient to prohibit casting induced atrophy, demonstrating that these proteases play important roles in disuse atrophy (123) Finally, we report that caspase 9, a caspase that can lead to the activation of caspase 3 via a mitochondria mediated pathway (69) is also activated in the plantaris muscle by increased mitochondrial ROS emission during prolonged disuse. In contrast, we did not observe inactivity induced activation of caspase 9 in the soleus muscle. The explanation for this muscl e specific difference is unclear but may be linked to the fiber type differences between these two mu scles or the fact that the time course of caspase 3 activation during inactivity differs between these two muscles. Much emphasis has been placed on the ro le that the proteasome system plays in disuse muscle atrophy. Importantly, these experiments reveal for the first time that increases in mitochondrial ROS emission are required for casting induced increases in the muscle specific E3 ligases atrogin 1 and M uRF 1. Increases in these E3 ligases are commonly associated with increase d FoxO3a and NF (58) Nonetheless, we did not detect activation of NF kB or FoxO3a following immobilization in either the soleus or plantaris muscles. Recent evidence suggests that increased autophagy contributes to different forms of skeletal muscle atrophy including atrophy induced by denervation (19, 38, 52, 70, 86) Further, accumulating evidence suggests that oxidative stress plays an important role in the induction of autophagy (reviewed in (67) ). In this regard, our results reveal that inactivity induced increases in autophagy signaling in the plantaris and

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44 soleus muscles can be prevented by treatment of animals with the mitochon drial targeted antioxidant SS 31. Finally, we hypothesized that the increase in mitochondrial ROS emission from limb muscles during casting promotes a decrease in anabolic signaling (i.e., Akt/mTor signaling) which can decrease protein synthesis and accele rate protein degradation by activation of both the proteasome and autophagy proteolytic systems. Our data support this prediction and reveal that increased mitochondrial ROS plays an important role in the inactivity induced decrease in Akt and mTor phospho rylation. Future experiments will be required to determine if prevention of disuse induced decreases in anabolic signaling can prevent inactivity mediated decreases in muscle protein synthesis. Conclusions Our findings clearly demonstrate that increased mi tochondrial emission of reactive oxygen species are important contributors to disuse muscle atrophy. Inactivity induced activation of all four key proteolytic systems in both the soleus and plantaris muscles was prevented by treatment with a mitochondria t argeted antioxidant (i.e.,SS 31) Finally, treatment with a mitochondrial targeted antioxidant also prevented the disuse induced decrease in anabolic signaling (i.e., Akt/mTor ) in the casted skeletal muscles suggesting that increased mitochondrial ROS emis sion acts to depress the Akt/mTOR pathway in skeletal muscles undergoing disuse induced atrophy.

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45 Figure 3 1. Experimental Design to address Aim 1

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46 A) B) Figure 3 2. Reactive oxygen species emission. Hydrogen peroxide (H2O2) release from permeabilized A) soleus and B) plantaris muscle fibers. V alues are presented as fold control of mean arbitrary units SEM. = significantly different from all other groups (p<0.05). from Con and Con SS ( p < .05).

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47 A) B) C) CON CON SS CAST CAST SS Soleus Plantaris Figure 3 3. Cross sectional area and representative images. Muscle fiber cross sectional area ( CSA ) by fiber type for the A) soleus and B) pla ntaris. Values are mean SEM. = significantly different from all other groups ( p < .05). C) Representative staining of MHC I ( blue ), MHC IIa ( green ), and dystrophin ( red ) proteins

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48 Table 3 1. Mitochondrial f unct ion of permeabilized soleus fiber b undles. Parameter CON CAST CAST SS State 3 VO 2 5.401.4 3.731.1 5.701.5 State 4 VO 2 1.240.3 1.880.5* 1.170.2 RCR 4.451.2 2.171.1* 4.761.5 State 3 and State 4 values are mean oxygen consu m ption, normalized to fiber bundle different from CAST SS = significantly different from all other groups (p<0.05). Table 3 2. Mitochondrial f unction of permeabilized plantaris fiber b un dles. Parameter CON CAST CAST SS State 3 VO 2 7.640.60 8.781.81 State 4 VO 2 1.200.29 1.640.59 1.670.30 RCR 7.082.37 5.511.03 State 3 and State 4 values are mean oxygen consu m pt ion, normalized to fiber bundle dry weight, different from CAST = significantly different from CON (p<0.05).

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49 SOLEUS PLANTARIS A) B) C) D) Figure 3 4. Calpain activation and activity. Levels of active calpain (A soleus, and B, plantaris ) were assessed via Western blot. Calpain activity (C, soleus, and D, plantaris II spectrin cleavage product of calpain (145 kD). Representative Western blots are shown below the graphs. Values are presented as fold control of mean tubulin, which is shown under the representative blot = significantly different vs. all other groups ( p p < .05).

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50 SOLEUS PLANTARIS A) B) C) D) E ) F) Figure 3 5. Caspase 3 activation and activity; caspase 9 activation Levels of active caspase 3 (A soleus, and B, plantaris ) were as sessed via Western blot. Caspase 3 activity (C, soleus, and B, plantaris ) was assessed by measuring l II spectrin cleav age product of caspase 3 (120 kD). Caspase 9 activation, an upstream activator of caspase 3, was also assessed (E, soleus, and F, plantaris). Values are fold control of mean arbitrary units SEM. Blots were normali tubulin. = significantly different vs. all other groups ( p p < .05).

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51 SOLEUS PLANTARIS A) B) C ) D ) Figure 3 6. E3 Ligase expres sion. mRNA expression of Atrogin 1 (A, soleus, and B, plantaris) and MuRF 1 (C, soleus, and D, plantaris) was assessed. Values are fold control of mean arbitrary units SEM. *= significantly different from all other groups ( p erent vs. Con ( p < .05).

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52 SOLEUS PLANTARIS A ) B ) C) D) Figure 3 7. Markers of autophagy. The ratio of LC3 II/I (A, soleus, and B, plantaris) was assessed as an index of autophagosome formation. Cathepsin L mRNA expression was also assessed (C, soleus, and D, plantaris) Values are presented as fold control of different vs. Con ( p < .05).

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53 SOLEUS PLANTARIS A) B) Figure 3 8. PGC PGC B, plantaris ). Values are presented as fold control of mean arbitrary units SEM p < .05).

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54 SOLEUS PLANTARIS A) B) C ) D) Figure 3 9. Markers of anabolic signaling. The ratio of pAkt/Akt (A, soleus, and B, plantaris) and pmTOR/ mTOR (C, soleus, D, plantaris) was assessed. Values are presented as fold control of mean arbitrary units SEM. Blots were tubulin, which is shown under the representative blot = significantly different vs. all other groups ( p < .05). vs. Con ( p < .05).

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55 CHAPTER 4 CALPAIN AND CAPASE 3 PLAY REQUIRED ROLE S IN IMMOBILIZATION INDUCED LIMB MUSCLE ATROPHY 1 Prolonged periods of skeletal muscle inactivity due to space flight, bed rest, or limb immobilization result in muscle atrophy in both animals and humans. Due to both the invasive nature of human muscle biopsies and the desire to use pharmacological interventions not approved for humans, animal models are commonly used to explore the mechanisms responsible for disus e atrophy, with hindlimb immobilization (i.e., casting) of rodents often used to mimic human limb immobilization Disuse skeletal muscle atrophy results from both decreased protein synthesis and increased protein degradation, with degradation playing a ma jor role (124) Four major proteolytic systems (autophagy, the ubiquitin protease system (UPS), calpains, and caspase 3) are active during atrophy in skele tal muscle (95) However, the degree to which each system contributes to disuse atrophy remains debatable. It has been argued that the UPS plays a dominant role during inactivity induced muscle atrophy because the UPS is responsible for degrading most sarcomeric proteins (47, 80) Nonetheless, the UPS cannot degrade intact actin and myosin because of the closely packed arrangement of these proteins in the sarcomeres of striated muscle (30, 119) Therefore, the UPS may not be the rate limiting step in muscle atrophy, as release of myofilaments could be a prerequisite for proteasome mediated degradation of both actin and myosin. Calpain and caspase 3 are two proteases that may assist the UPS by releasing II 1 Reprinted with permission from Journal of Applied Physiology: Talbert EE, Smuder AJ, Min K, Kwon OS, and Powers SK. Calpain and capase 3 play required roles in immobilization induced limb muscle atrophy. J Appl Physiol 2013.

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56 fodrin), titin, and nebulin (50) Breakdown of these cytoskeletal proteins releases actin and my osin from sarcomeres, allowing muscle specific E3 ligases to ubiquitinate myofilament proteins, targeting them for degradation by the UPS. Further, caspase 3 has been reported to degrade intact actomyosin (30) Nonetheless, definitive evidence that calpain and/or caspase 3 play a required role in limb disuse muscle atrophy does not currently exist. Indeed, disagreement exists regarding the role that calpain plays in disuse induced atrophy in limb muscles (102, 125) and incomplete data is a vailable regarding the part that caspase 3 plays in limb muscle atrophy (90) Recent work in mechanical ventilation induced diaphragm atrophy suggests that 3 in respiratory muscles. Indeed, inhibition of calpain prevented caspase 3 activation in the diaphragm during mechanical ventilation, suggestin g that calpain regulates caspase 3 activity. Caspase 3 inhibition also prevented calpain activation, demonstrating caspase 3 regulation of calpain activity (85) Nevertheless, mechanical ventilation induced diaph ragmatic atrophy is an unusually rapid and unique type of muscle atrophy, and it remains unclear if this same regulatory crosstalk between calpain and caspase 3 exists in limb skeletal muscles during immobilization induced atrophy (51, 68) Therefore, because of limited knowledge regarding the role that calpain and caspase 3 play in disuse atrophy in limb skeletal muscles, these experiments were designed to achieve two goals: 1) to determine if inhibition of calpain or caspase 3 is sufficient to protect against immobilization induced limb muscle atrophy, and 2) to determine if a regulatory cross talk exists between calpain and caspase 3 in limb skeletal m uscle. We hypothesized that both calpain and caspase 3 play important roles

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57 in disuse limb muscle atrophy and that a regulatory cross talk exists between these two proteases. Our findings support these hypotheses and demonstrate for the first time that bot h calpain and caspase 3 play a required role in inactivity induced atrophy of limb muscle. Further, our results also reveal that a calpain/caspase 3 regulatory cross talk exists in limb skeletal muscle whereby calpain can promote caspase 3 activation and a ctive caspase 3 can induce calpain activation in the soleus muscle during prolonged immobilization. Methods Animals and Experimental Design Animal groups. Female 300g Sprague Dawley rats (Charles River, Wilmington, MA) were assigned to one of 6 groups (Fi gure 4 1) : 1) non casted receiving saline injections (Con, n=10), 2) non casted receiving the calpain inhibitor, SJA 6017 (Con Calp, n=6), 3) non casted receiving the caspase 3 inhibitor Ac DEVD CHO (Con Cas 3, n=10), 4) hindlimbs casted receiving saline ( Cast, n=8), 5) hindlimbs casted receiving the calpain inhibitor, SJA 6017 (Cast Calp, n=7), and 6) casted receiving the caspase 3 inhibitor Ac DEVD CHO (Cast Cas 3, n=8). All experiments were approved by the University of Florida Institutional Animal Care and Use Committee. Immobilization. Gaseous isoflurane (2% for induction of anesthesia, 0.5 1.5% to maintain a surgical plane of anesthesia) was used during casting. After reaching a it (3M Health Car e, St. Paul, MN) was applied to the entire area to be casted to prevent skin abrasions. A plaster of Paris cast (Gypsona, Reynosa, Tamaulipas, Mexico) was applied from the last rib down through both legs with the ankle plantar flexed as to induce maximum s oleus atrophy. To prevent casted animals from damaging the cast by chewing, a single layer of

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58 of Paris. Animals were allowed to recover from anesthesia and monitored carefully to ensure unhindered access to food and water within their cages. Animals were removed from their cages daily to check for any damage to the cast or swelling or abrasions to the feet. Pharmacological Inhibitors Calpain Inhibition To inhibit calpain activity in the soleus muscle during hindlimb immobilization, we admin istered SJA 6017 (Calpain inhibitor IV, EMD Chemicals, Gibbstown, NJ) at a dose of 3 mg/kg body weight/day. SJA 6017 was dis solved in 88% propylene, 10% ethyl alcohol, 2% benzyl alcohol. Anima ls were injected subcutaneously immediately prior to casting and then every 24 hours for seven days. Caspase 3 Inhibition To inhibit caspase 3 activity during hindlimb immobilization, we admin istered the caspase 3 inhibitor AC DEVD CHO (Enzo Life Science s, Farmingdale, NY) at a dose of 3 mg/kg body weight/day. AC DEVD CHO was dissolved in 0.9% ster ile saline. Animals were injected subcutaneously in the neck immediately prior to casting and then every 24 hours for seven days. Specificity of calpain and ca spase 3 inhibition. Our laboratory has previously performed experiments inhibiting calpain activity in skeletal muscle using SJA 6017 and inhibition of caspase 3 with Ac DEVD CHO. In this regard, these in vitro experiments demonstrate that these inhibitors target only the intended protease without off target effects (85) More specifically, the calpain inhibitor SJA 6017 does not inhibit the activity of either caspase 3, or caspase 9, a caspase responsible for the activation of caspase 3. Further, these data show that AC DEVD CHO does not inhibit the activity of calpain 1 or calpain 2 (85)

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59 Antioxidant properties of the inhibitors. Because oxidative stress can promote disuse atrophy (97, 98) we used a Trolox Equivalent Antioxidant Capacity assay (99) to determine if either inhibitor possessed antioxidant properties. Our results reveal that neither protease inhibitor (e.g., S JA 6017 or AC DEVD CHO) quenched the oxidants used in the assay (data not shown). Tissue Harvesting At the completion of the experimental period, animals were again anesthetized using isoflurane. Once animals reached a surgical plane of anesthesia, both solei were removed. One soleus was divided in half with one half frozen for cross sectional area analysis and the other permeabilized to measure mitochondrial function. The remaining soleus was snap frozen in liquid nitrogen and saved for Western blotting and RT PCR analysis. Tissue was stored at 80C until analysis. After tissue removal, animals were sacrificed by removal of the heart. Biochemical Measures Preparation of permeabilized muscle fibers. We measured mitochondrial oxygen consumption in permeabi lized muscle fibers as previously described (79) Briefly, ~10 mg pieces of soleus muscle were teased ap art on ice in cold buffer X (60 mM K MES, 35 mM KCl, 7.23 mM K 2 EGTA, 2.77 mM CaK 2 EGTA, 20 mM imidazole, 0.5 mM DTT, 20 mM taurine, 5.7 mM ATP, 15 mM phosphocreatine, and 6.56 mM MgCl 2 pH 7.1). After dissection, soleus fiber bundles were rotated in Buffer saponin for 30 min at 4C to permeabilize the fiber membranes. The permeabilized bundles were washed three times for 5 minutes by rotation in buffer Z (110 mM K MES, 35 mM KCl, 1 mM EGTA, 5 mM K 2 HPO 4 3 mM MgCl 2 0.05 mM glutamate, 0. 02 mM malate, and 0.5 mg/ml BSA, pH 7.1).

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60 Measurement of mitochondrial function. Oxygen consumption by mitochondria in permeabilized soleus fibers was determined as previously described (79) Mitochondria respired on 5 mM pyruvate and 2 mM malate. State 3 respirati on was stimulated by stimulate state 4 respiration. The respiratory control ratio (RCR) was computed as the ratio of state 3 oxygen consumption to state 4 oxygen consumption. Myofib er cross s ectional a rea Pieces of soleus muscle were frozen in Optimal Cutting Temperature (OCT) medium in liquid nitrogen cooled isopentane, as previously described (74) ome (Shandon, Pittsburgh, PA) and stained for dystrophin and myosin heavy chain isoforms as described previously (74) Fiber cross sectional area was determined by tracing membrane boundaries using Scion Image software ( Scion Corp., Frederick MD). At least 100 fibers were analyzed for each animal. Western b lotting Soleus muscles were homogenized using a motorized glass on glass technique with 1:10 (mg wt/l buffer) 5 mM Tris/5 mM EDTA (pH 7.5) containing protease inhibit or cocktail 1:20 (vol/vol) (Sigma Aldrich). Following homogenization, samples were centrifuged at 1,500 g for 10 min at 4C. Protein concentration in the supernatant was determined using the method of Bradford (17) mercaptoethanol in Laemmli buf fer (Bio Rad, Hercules, CA). Membranes were probed for cleaved (active) calpain 1, cleaved caspase 3 (Cell Signaling, Danvers, MA), Bid/tBid (Imgenex, San Diego, CA), total cal II spectrin (Santa Cruz Biotechnology, Santa Cruz, CA) Images were visualized using the Odyssey system (Licor Biosciences,

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61 Lincoln, NE). To control for protein loading and transfer differences, each protein was tubulin (Santa Cruz) detected on the same membrane. Real t ime revers e t ranscriptase p olymerase c hain r eaction. Messenger RNA was isolated and cDNA synthesized as previously described (117) Briefly, TRIzol reagent (Life Technologies, Carlsbad, CA) was used according to the manufacturer's directions to isolate total RNA. RNA content (g/ muscle) was determined by spectrophotometry. cDNA was produced using th e Superscript II First Strand Synthesis System (Life Technologies). Three g RNA was reverse protocol. Taqman chemistry (Applied Biosystems, Foster City, CA) was used to determine the relative expression of calpastatin. The ABI Prism 7000 Sequence Detection system (Applied Biosystems) was used to perform the computed tomography method (Applied Biosystems, User Bulletin no. 2, ABI PRISM 7700 Sequence Detection System). Twenty used to determine calpastatin expression (Entrez Gene ID 25403 ) and expression of the calibrator sample glucuronidase [ Entrez Gene ID 244434]) glucuronidase a lysosomal glycoside hydrolase, was chosen as the refere nce gene based on previous work showing unchanged expression in another model of disuse atrophy, mechanical ventilation (26, 27) Statistical a nalysis For cross sectional area, comparisons between groups were made u sing two way ANOVA with Bonferroni post tests used to determine differences in muscle fiber size between casted/non casted animals for each of the 3 treatment groups. For the rest of the analysis, comparisons between groups for each dependent

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62 variable were made by a one way analysis of variance and, when ap honestly significant differ ence test was performed post hoc Significance was established at p < .05. Data are presented as means standard error of the mean. Results Independent I n hibition of C alpain and Caspase 3 P revents Casting induced M uscle A trophy These experiments used a rat model of hindlimb immobilization to mimic the atrophy that occurs in humans during the casting of a limb. Analysis of the cross sectional area (CSA) of Type 1 soleus fibers by two way ANOVA revealed that an interaction effect exists between our casted/non casted and drug treatment variables following seven days of treatment. A Bonferroni post test concluded that casting only had a significant effect in th e saline treated group ( Figure 4 2 A). In contrast, casting did not induce significant changes in the CSA of animals treated with either our calpain inhibitor (SJA 6017) or our caspase 3 inhibitor (AC DEVD CHO), demonstrating that both calpain and caspase 3 play important roles in inactivity induced muscle atrophy. An experimental concern with pharmacological inhibition of proteases is that prolonged protease inhibition will induce muscle hypertrophy, potentially masking atrophy. To determine if our protease ambulatory animals with either the calpain or caspase 3 inhibitor for 7 days. We found neither an interaction effect nor main effects when analyzing our Type IIA CSAs ( Figure 4 2B ), demonstratin g that no significant changes occurred in Type IIA soleus fibers in response to either daily treatment with either of our protease inhibitors or casting Representative muscle cross sections of all experimental groups appear in Figure 4 2 C H.

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63 Casting Activ ates C alpain in the Soleus during 7 days of I mmobilization Casting significantly increased the presence of the cleaved (active) band of calpain 1 following 7 d ays of cast immobilization (Figure 4 3 A). Casting also significantly increased the presence of a calpain II spectrin that II spectrin ( Figure 4 3B ). This calpain specific II spectrin is an index of in vivo calpain activity (129) Daily treatment with the calpain inhibitor SJA 6017 prevented the activation of calpain 1 and the increase in the calpain II spectrin fragment, demonstrating that our inhibition of calpain activation in the soleus muscle was successful. Interestingly, independent inhibition of caspase 3 also prevented activation of calpain in the immobilized soleus muscle. The prevention of calpain activation by inhibition of caspase 3 demonstrates that caspase 3 plays a regulatory role in the activation of calpain. Casting A ctivates C aspase 3 in the S oleus during 7 days of I mmobilization Seven days of cast immobilization a lso significantly activated caspase 3 in the soleus muscle, demonstrated by an increase in cleaved (active) caspase 3 ( Figure 4 3C ). Casting also significantly increased the caspase II II spectrin that can be detected at 120 kD on a Western blot ( Figure 4 3D ). Importantly, treatment of animals with the caspase 3 inhibitor Ac DEVD CHO blunted the immobilization induced activation of caspase 3 and completely prevented the increase in the 120 kD produ II spectrin in the soleus muscle. Calpain inhibition also prevented the full activation of caspase 3 and completely prevented the increase in the caspase 3 II spectrin fragment, demonstrating that calpain plays a regulatory role in the a ctivation of caspase 3. Together, these

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64 results indicate that a regulatory cross talk exists between the calpain and caspase 3 in the soleus muscle during immobilization induced fiber atrophy. Potential M echanism(s) R esponsible for R egulatory C ross talk b etween C alpain and C aspase 3 Following the observation that regulatory cross talk exists between calpain and caspase 3, we sought to determine the mechanism by which active calpain promotes caspase 3 activation. Evidence in non muscle cells indicates that calpain can cleave Bid, a pro apoptotic protein, to truncated Bid (tBid). tBid then translocates to the mitochondria, leading to the release of cytochrome c followed by caspase 3 activation (22) Similar results have been reported in diaphrag m muscle during prolonged mechanical ventilation (85) Our results reveal that immobilization of the soleus muscle results in an increase in both tBid and the tBid/Bid ratio ( Figure 4 4 A C). The change in tBid/Bid ratio was driven by an increase in tBid, as all three casted groups demonstrated no differences in the abundance of Bid ( Figure 4 4B ). Importantly, this in activity induced increase in the tBid/Bid ratio was blunted by inhibition of calpain activity in the soleus muscle ( Figure 4 4C ). Although these observations do not provide definitive proof, our results are consistent with the notion that the calpain media ted activation of caspase 3 can occur, in part, by the formation of the pro apoptotic protein tBid. This is a testable hypothesis worthy of further research. We then sought to determine the mechanism by which active caspase 3 promotes the activation of ca lpain. Calpastatin, the endogenous inhibitor of calpain, is a known substrate of caspase 3. Active caspase 3 may degrade calpastatin, removing (130) Our results reveal that calpastatin levels decreased in the soleus muscle during prolonged immobilization ( Figure 4 5A ). The

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65 decline in calpastatin levels de creased the calpastatin/calpain ratio, which is an index of the ability of calpastatin to inhibit calpain. Inhibition of caspase 3 activity led to calpastatin levels statistically similar to both control and casted animals ( Figure 4 5A ). Total calpain expr ession was not different between any of the groups ( Figure 4 5B ). Similarly to calpastatin expression, the calpastatin/calpain ratio in the soleus muscle of casted animals treated with our caspase 3 inhibitor was similar to both control and casted animals ( Figure 4 5C ). Hence, our results do not provide firm evidence that caspase 3 regulates calpain activity by degrading calpastatin in limb muscles during inactivity. To ensure that casting simply was not increasing the turnover of calpastatin, we measured the mRNA expression of calpastatin, Calpastatin mRNA was decreased by casting and not rescued by either protease inhibitor ( Figure 4 5 D). Inhibition of both C alpain and C aspase 3 P rotect against I mmobilization induced M itochondrial D ysfunction No significa nt differences in oxygen consumption between groups were found during active state 3 respiration or basal state 4 respiration. However, hindlimb casting resulted in a ~50% decrease in the respiratory control ratio of mitochondria in permeabilized fibers. I ndependent treatment of animals with a calpain or caspase 3 inhibitor partially prevented this decrease in mitochondrial coupling, with calpain inhibition providing a strong trend towards complete protection (p=0.056) (Table 1). Discussion Overview of Maj or Findings This is the first investigation to demonstrate that independent inhibition of calpain or caspase 3 in locomotor skeletal muscle is sufficient to prevent immobilization induced Type I fiber atrophy. Further, our findings also reveal that regulat ory cross talk

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66 exists in limb skeletal muscle whereby calpain and caspase 3 can activate one another. Finally, these results also provide new information about the impact of calpain and caspase 3 activation on mitochondrial function in locomotor skeletal m uscle. Independent I nhibition of C alpain or C aspase 3 A ctivity P revents I mmobilization induced S oleus M uscle A trophy Similar to previous reports in diaphragm muscle (72, 74, 85) the current study reveals that independent pharmacological inhibition of calpain or caspase 3 is sufficient to prevent disuse muscle atrophy. However, note that our results indicate that 7 days of limb immobilization promoted atrophy in Type I fibers only as this duration of muscle disuse was not sufficient to achieve significant atrophy in the Type I Ia fibers within the soleus muscle. Nonetheless, in the adult Sprague Dawley rat, ~85% of the soleus fibers are Type I (49) and therefore, our observed protection against disuse fiber atrophy is physiologically important. Differences exist in the level of protection against dis use muscle atrophy between the current study which used a highly selective pharmacological inhibitor of calpain and previous reports using the overexpression of calpastatin to retard calpain activity. The two published reports using calpastatin overexpress ion to prevent calpain activation reported only partial (125) or minimal (102) protection against hindlimb suspension induced muscle atrophy. The explanation for these divergent findings is unclear but could be due to several factors. First, the duration of muscle inactivity and the experimental model of muscle disuse differed between the current study and these earlier studies. Indeed, the present study used 7 days of immobilization to induce muscle atrophy whereas 10 14 days of hindlimb suspension was utilized in both of the calpastatin overexpression studies. These studies using hindlimb suspension saw

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67 greater atrophy than our casting model, which may play a role in the level or type of protease activation. Secondly, neither of the calpastatin overexpression investigations evaluated muscle atrophy by assessing fiber type specific cross sectional areas which may account for some of the differences between the c urrent study and these previous reports. Additionally, the current study utilized a rat experimental model, while both calpastatin studies used a murine model to investigate disuse muscle atrophy. Finally, the study of Tidball and Spencer (125) acknowledges that they did not achieve complete inhibition of calpain activity in skeletal muscle, where as our data (Figure 4 3 B, II spectrin levels) indicates that the calpain activity in the soleus muscle of our casted animals receiving the calpain inhibitor did not differ from the calpain activity in the soleus muscle of an ambulatory control animal. I mportantly, our results also reveal that pharmacological inhibition of caspase 3 provides protection against immobilization induced muscle atrophy in Type I fibers of the soleus muscle. Similar results have been reported in ventilator induced atrophy of di aphragm muscle (74, 85) Moreover, a recent report also concluded that denervation induced gastrocnemius muscle atrophy is diminished in caspase 3 knockout mice (90) However, the present experiment reveals that caspase 3 inhibition results in compl ete protection against disuse induced soleus muscle atrophy whereas Plant et al. (90) report only a mode st reduction in gastrocnemius muscle atrophy in the wild type vs. the caspase 3 knockout mice. The explanation for these divergent findings is unclear but species differences, duration of experiment, fiber type differences between gastrocnemius and soleus muscles, and/or differences in atrophy model (i.e., denervation vs. immobilization) could be responsible for these disparities.

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68 Regulatory C ross talk E xists between C alpain and C aspase 3 Our data provide the first evidence that a regulatory cross talk e xists between calpain and caspase 3, as inhibition of calpain is sufficient to prevent activation of caspase 3 in limb skeletal muscle. Similarly, inhibition of caspase 3 prevented calpain activation in the inactive soleus muscle. To investigate the mechan ism responsible for these findings, we tested the hypothesis that calpain can potentially activate caspase 3 through cleavage of Bid to form tBid. tBid is a membrane pore forming protein that can release cytochrome c from the mitochondria resulting in the subsequent activation of caspase 3 (22) Our results reveal that inhibition of calpain activity results in a significant attenuation of the immob ilization induced increase in tBid/Bid rati o in the soleus muscle (Figure 4 4 C). Thus, it is feasible that calpain activates caspase 3 in skeletal muscle disuse atrophy via a tBid dependent mechanism. Nonetheless, this evidence is not definitive as active caspase 3 can also lead to Bid cleavage, leading to a feed forward activation of caspase 3 (115) Therefore, additional work will be required to determine the specific mechanism(s) by which active calpain promotes caspase 3 activation. To investigate the mechanism responsible for the caspas e 3 mediated activation of calpain, we measured the protein abundance of both calpain and the endogenous calpain inhibitor, calpastatin. In this regard, our prior work revealed that caspase 3 inhibition prevented the decrease in calpastatin and the calpast atin calpain ratio associated with mechanical ventilation induced diaphragm atrophy (85) The current experiments also demonstrate that caspase 3 inhibition blunts the casting induced decrease in calpastatin leve ls in the soleus muscle (Figure 4 5 A), and either calpain inhibition and caspase 3 inhibition are sufficient to provide very modest protection

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69 against immobilization induced decreases in the calpastatin/calpain ratio (Figure 4 5 C). Nonetheless, because the inhibition of caspase 3 does not completely prevent the immobilization induced decrease in both calpastatin and the calpastatin/calpain ratio in the soleus muscle, it is possible that caspase 3 mediated activation of calpain may involve other mechanisms i n skeletal muscle exposed to long periods of inactivity. An important difference between the current study and our previous work in diaphragm muscle is the duration of muscle inactivity. Indeed, Nelson et al. (85) investigated mechanical ventilation induced diaphragm atrophy following only 12 hours of inactivity while the current study investigated limb muscle atrophy follo wing 7 days of immobilization. Thus, caspase 3 inhibition may be sufficient to prevent calpastatin degradation in skeletal muscle in the short term but this may not be an important protective mechanism over longer time periods. Supporting this prediction i s the observation that a decrease in calpastatin gene expression following 7 days of casting was not rescued by either calpain or caspase 3 inhibition ( Figure 4 5 D). Protection of Mitochondrial Function by Protease Inhibition Prolonged muscle inactivity is associated with mitochondrial dysfunction in both limb and diaphragm muscle (60, 79, 92) Consistent with previous findings the current investigation shows that 7 days of immobilization induced a significa nt decrease in the respiratory control ratio (RCR) of mitochondria in permeabilized soleus fibers (Table 1). RCR is an index of mitochondrial coupling and a decrease in RCR indicates that mitochondria in the casted muscle are less coupled than mitochondria within the soleus muscles of weight bearing animals. Interestingly, caspase 3 inhibition prevented a significant decline in mitochondrial function and inhibition of calpain activity in the soleus muscle almost completely rescued the immobilization induced decline in the

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70 mitochondrial RCR. Although our experiments do not provide a mechanism to explain this observation, one potential explanation is that calpain 10, a mitochondrial calpain linked to mitochondrial dysfunction (8) might be inhibited by our calpain inhibitor. If this is the case, prevention of calpain 10 activation could protect mitochondrial electron transport chain proteins from degradation and thus, preserve mitochondrial function. This is another testable hypothesis worthy of future experimentation. Conclusions In conclusion, this study revealed that inhibition of either calpain or caspase 3 is sufficient to prevent casting induced atrophy of the soleus, prov iding evidence that both calpain and caspase 3 are important proteases in disuse limb muscle atrophy. Further, we have demonstrated for the first time in limb muscle that a regulatory crosstalk exists between calpain and caspase 3 whereby calpain can stimu late caspase 3 activation and active caspase 3 can promote calpain activation. Finally, our results reveal that active calpain and to a lesser extent, active caspase 3 can promote mitochondrial uncoupling in skeletal muscle. The mechanism(s) responsible fo r this finding is unknown and warrants further research.

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71 Figure 4 1. Experimental Design to address Aim 2

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72 A) B) C) D) E) F) J) H) Figure 4 2. Cross sectional area and representative images. Soleus muscle fiber cross sectional area ( CSA ) by fiber type. A) Quantification of Type I myofibers. Values are meanSEM. p < .05). B) Quantification of Ty pe IIa myofibers. Values are meanSEM. C) Representative staining of MHC I ( blue ), MHC IIa ( green ), and dystrophin ( red ) proteins in a control cross section. D) Representative staining of Con Calp. E) Representative staining of Con Cas 3. F) Representative staining of Cast. G) Representative staining of Cast Calp. H) Representative staining of Cast Cas 3. Scale bars =

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73 Figure 4 3. Activation and activity of calpain and caspase 3. Activation and activity of the proteases calpain 1 ( A B ) and caspase 3 ( C D ) were determined via Western blotting. A) Active calpain 1 II spectrin cleavage product of calpain (145 kD), C) Active caspase II spectrin cleavage product of caspase 3 (120 kD). Values are presented as fold control tubulin = significantly different vs. all other groups ( p vs. Con ( p < .05).

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74 A ) B) C) Figure 4 4. Expression of Bid, tBid, and the ratio of tBid/Bid. t Bid, Bid, and the tBid/Bid ratio. A) tBid expression B) Bid expression C) tBid/Bid ratio. Values are mean arbitrary units SEM. *= significantly different from all other groups ( p < .05). p < .05). and Cast ( p < .05).

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75 A) B) C) D) Figure 4 5. Expression of Calpastatin and Total Calpain. A) Calpastatin expression, B)Total calpain expression, C) Calpastatin/calpain. Representative images appear above Significantly different vs. Con (p < .05).

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76 Table 4 1. Mitochondrial f unction. Parameter Con Cast Cast Calp Cast Cas 3 State 3 VO 2 5.401.43 3.731.09 4.881.09 5.101.26 State 4 VO 2 1.240.30 1.880.52 1.520.63 1.530.36 RCR 4.451.18 4.071.90 a 3.3 9 0.68 Oxygen consumption from permeabilized soleus fibers was determined during both State 3 (ADP stimulated) and State 4 (following ADP stimulation). The respiratory control ratio (RCR) is the ratio of these two values and is a measure of mitochondrial coupli S ignificantly different vs. Con (p < .05). a = Note that a trend towards significant differences exists between Cast and Cast Calp, p=0.056.

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77 CHAPTER 5 BLOCKAGE OF THE RYAN ODINE RECEPTOR DURIN G MECHANICAL VENTILATION IS NOT S UFFICIENT TO PREVENT ATROPHY OR INCREASED MITOCHONDRIAL REACTI VE OXYGEN SPECIES PR ODUCTION Mechanical ventilation (MV) is a method of sustaining pulmonary gas exchange in patients who are unable to maintain adequate alveolar ventilation on their own. While MV is a life saving critical care measure, extended periods of MV lead to diaphragm atrophy and contractile dysfunction ( 52, 68, 89) collectively termed ventilator induced diaphragm dysfunction (VI DD). This MV induced diaphragm weakness often leads to an inability to wean critical care patients from ventilators, leading to lengthy hospital stays and increases in morbidity and mortality. Approximately 25% of intensive care patients experience difficult weaning (33) so MV induced inspiratory muscle weakness is an important clinical problem. I ncreases in reactive oxygen species (ROS) pr oduction in the diaphragm are required for VIDD (10, 114, 133) The major source of ROS during prolonged MV appears to be the mitochondria (60, 92) and this increase in mitochondrial ROS emission activates the proteases calpain and caspase 3 which are important contributors to the development of VIDD (92) In addition to an increase in ROS production, prolonged MV also likely increases calcium levels in the cytosol of diaphragm muscle fibers (53, 59) and this increa se in cytosolic calcium is a requirement for the activation of calp ain (57) The sources of this increase in intracellular calcium remain unknown. However, o ne potential source of increased cytosolic calcium in the diaphragm during prolonged MV is leakage of calcium into the cytosol through the ryanodine receptor (RyR). The RyR is the gated calcium channel of the sarcoplasmic reticulum ( SR) (36) which generally remains closed unless

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78 stimulated to open by a nerve impulse leading to calcium release from the SR and a muscle contraction. However, oxidation of the RyR can result in calcium leak into the cytosol (4) Generally, increases in cytosolic calcium lead to increases in calcium content of the mitochondrial matrix because of the charge differential between the cytosol and mitochondrial matrix (87) Increased mitochondrial Ca 2+ content stimula tes several Kreb s cycle enzymes and increas es electron flow through the electron transport chain (ETC) (41, 110) During periods of muscle contractions, increased mitochondrial calcium increases mitochondrial production of ATP However, during prolonged inactivity additional energy production is not required, and no ADP is available to phosphorylate. Because the potential across the mitochondrial membrane is not being used to produce ATP the energy potential continue s to rise (110) and can lead to increas e d electron leak and elevated mitochondrial ROS production (66, 81) In additio n to increased mitochondrial ROS emission, prolonged MV is also associated with decreases in diaphragm mitochondrial coupling (60, 79, 92, 118) and increases in mitochondrial calcium could contribute to this dysfunction. Specifically, large increases in mitochondrial calcium uptake can lead to formation and opening of the mitochondrial permeability transition pore (20) The opening of the transition pore results in the loss of the mitochondrial membrane potential, and leads to a decrease in ETC flux and energy production, along with swelling of the mitochondria (46) Opening of the transition pore can also lead to the activation of caspase 3 (45) a required protease for the development of VIDD.

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79 In regard to the contribution of the RyR to MV induced alterations in cytosolic calcium levels in the diaphragm, r ecent evidence suggests that leaky RyRs contribute t o age induced sarcopenia (4) muscle dystrophy (5) and skeletal muscle dysfunction due to heart failure (131) Muscle function in animal models of these conditions is improved by treatment with a compound that stabilizes the RyR (4) Further, work by Fischer et. al. demonstrated that treatment with dantrolene, a RyR blocking molecule, prevents sepsis indu ced calpain expression and muscle breakdown (37) Thus, we hypothesized that RyR leak may contribute to MV induced mitochondrial dysfunction and increased proteolytic activity (e.g., calpain and caspase 3). To test the hypothesis that RyR leak is responsible for MV induced increases in diaphragm mitochondrial ROS release, mitochondrial dysfunction, and atrophy, animals were treated with or without the pharmacological RyR blocker azumolene during 12 hours of M V. Our results reveal that blockade of the RyR is not sufficient to prevent MV induced diaphragm mitochondrial ROS production, protease activation, or diaphragmatic atrophy. Nonetheless, pharmacological inhibition of RyR is sufficient to protect against MV induced mitochondrial damage in the diaphragm. Methods Animals Young adult (300 g) female Sprague Dawley rats (Harlan Laboratories) were housed by University of Florida Animal Care Services. Animals received standard rat chow and water ad libitum. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the University of Florida Institutional Animal Care and Use Committee.

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80 Experimental Protocol Rats were randomly assigned to one of four group s (n=8 10, Figure 5 1) ; 1) Con Vehicle (Veh), 2) Con Azumolene (AZ), 3) MV Vehicle (MV) and 4) MV Azumolene (MVA) Animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg). Once animals reached a surgical plane of anes thesia, an incision was made in the neck, allowing access to the trachea, a carotid artery, and a jugular vein. The jugular vein was cannulated and either azumolene sodium (10 mg/kg) or vehicle (50% saline, 50% dimethylsulfoxide) was infused. Animals were then tracheostomized Immediately following tracheostomy animals in the Veh and AZ groups had both solei removed and then were sacrificed by removal of the heart and diaphragm. Animals in the MV and MVA groups underwent 12 hours of prolonged MV. These ani mals received either a constant infusion of vehicle (10% DMSO, 90% saline) or 0.83 mg/kg/hr sodium azumolene. The MV and MVA groups were sacrificed in the same manner at the conclusion of 12 hours. Details of MV Ventilator settings (Servo Ventilator 300, S iemens) were as follows: upper airway pressure limit: 20 cm H 2 O, typical pr essure generation above PEEP: 6 9 cm H 2 O, respiratory rate: 80 breaths/min ; and PEEP: 1 cm H 2 O). Animals were constantly monitored during MV. Continuing care included expressing the rotating the animals to prevent blood pooling, and suctioning the airway to prevent mucus plugs. At the start of MV, animals received an intramuscular injection of glycopyrrolate (0.08 mg/kg) to decrease airway mucus secretions during M V. Animals also received a maintenance dose of glycopyrrolate (0.04 mg/kg) every two hours intramuscularly. Body temperature was read by a rectal thermometer and maintained at

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81 37C by resting animals on a recirculating water blanket Heart rate was monitor ed by electrocardiograph. Following the initiation of MV, the carotid artery was cannulated to measure blood pressure. Blood s amples (~100 uL) were taken from this catheter to ensure blood gas homeostasis was maintained during MV. If blood gas parameters c hanged, either tidal volume or oxygen concentration of the inspired air was increased to return to homeostasis. To maintain a surgical plane of anesthesia, sodium pentobarbital diluted in heparinized saline was constantly infused into a catheter in the jug ular vein (~10 mg/kg/hr). Tissue Harvesting The diaphragm was removed and divided into several sections, including one to measure contractile function, one for mitochondrial measurements, and one stored for histochemistry. The remaining diaphragm was quic kly frozen in liquid nitrogen and stored at 80C for analysis by Western blotting One entire soleus was used to measure contractile function. Functional Measurements Contractile function: In vitro contractile function was measured in one entire soleus an d one ~25 mg strip of diaphragm. Due to concerns about the ability to find optimal length in animals treated with azumolene, soleus optimal length was set at 20 g of force, and diaphragm optimal length was set at 2 g. After 3 maximal tetanic contractions, an isometric force frequency curve was generated as previously described (96) Mitochondrial Measurements Permeabilization of diaphragm fiber bundles: Small strips of diaphragm were permeabilized according to the method of Anders on (3) Briefly, a ~10 mg pie ce of

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82 diaphragm was teased apart to near single fibers on ice in buffer X (60 mM K MES, 35 mM KCl, 7.23 mM K 2 EGTA, 2.77 mM CaK 2 EGTA, 20 mM imidazole, 0.5 mM DTT, 20 mM taurine, 5.7 mM ATP, 15 mM phosphocreatine, and 6.56 mM MgCl 2 pH 7.1). Fiber bundles we while rotating at 4C. Fiber bundles were then washed three times for five minutes each in buffer Z (110 mM K MES, 35 mM KCl, 1 mM EGTA, 5 mM K 2 HPO 4 3 mM MgCl 2 0.05 mM glutamate, 0.02 mM malate, and 0.5 mg/ml BSA, pH 7.1). Mitochondrial respiration: Mitochondrial oxygen consumption of a permeabilized fiber bundle was measured using previously described techniques (60) Both t he maximal oxygen consumption (ADP stimulated, state 3) and basal respiration (ADP limited, oligomycin stimulated, state 4) were measured The respiratory control ratio (RCR) an index of mitochondrial coupling, was calculated by dividing state 3 oxygen cons umpti on by state 4 (60) Oxygen consumption was normalized to the dry weight of the fiber bundle to control for differences in bundle size. Mitochondrial ROS emission: Diaphragmatic mitochondrial ROS emission from a permeabilized fiber bundle was determined using the Amplex Red TM r eagent (Life Technolo gies ). Details of this assay have been described previously (60) During this assay, diaphragm fiber bundles are incubated with succinate, creatine kinase, creatine phosphate, and creatine, which allows the mitochondria to respire and provides a more physiological measurement. Sample fluorescence was measured after 15 minutes of incubation at 37C. The fluorescence was normalized to dry weight of the tissue to control for bundle size.

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83 Histological Measure ments Cross sectional a rea: One piece of diaphragm was embedded in Optimal Cutting Temperature media (Sakura Fintek USA, Torrance, CA ) and frozen in li quid nitrogen using a c ryotome (ThermoScientific) and stained for dystrophin, myosin heavy chain (MHC) I and MHC type IIA proteins for fiber cross sectional area analysis (CSA) as described previously (75) CSA was determined using Scion Image software ( Scion Corp., Frederick MD ) Biochemical Measurements Tissue h omogenization, protein n ormalization, and W estern b lot a nalysis: Diaphragm tissue pieces were homogenized in 5 mM Tris, 5 mM EDT A buffer 10:1 (weight:volume) using a motorized glass on glass system. Protein concentration was determined using the Bradford method (17) and muscle samples were normalized to a standard protein conce ntration. Following normalization, abundance of specific proteins was determined in diaphragm samples via Western blotting using previously described methods (74) Protein abun tubulin, which served as a loading control. Electron Microscopy Approximately 1mm x 1mm squares of diaphragm were cut and fixed in 4% paraformaldehyde and 2.5% glutaraldehyde (pH 7.25) for one hour at room temperature. Samples w ere then stored at 4C until processing. Samples were prepared and imaged by the University of Florida ICBR Electron Microscopy Core Lab.

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84 Statistical Analysis Comparisons between groups for each dependent variable were made by a two way analysis of varianc e (ANOVA), using MV/con trol and vehicle/azumolene as the factors. When appropriate, a Bonferroni test was performed post hoc Comparisons of blood gas parameters were made using at two Significance was established a priori .05. Results Biological Response to MV No significant differences existed in body weight between the groups (Table 5 1). Treatment with AZ during prolonged MV did not cause any significant differences in arterial blood pressure, heart rate, Pa CO2 Pa O2 blood pH, or blood calcium concentration compared to MV animals not receiving the drug (Table 5 2). No animal exhibited any indication of infection or significant lung injury following 12 hours of MV. Impact of MV and AZ on Contractile Function To confirm the presence of azumolene within the muscle, we measured the in vitro specific force production in a strip of diaphragm muscle (Figure 5 2) and the intact soleus muscle (Figure 5 3) at stimulation frequen cies ranging from 15Hz to 160Hz. Azumolene administr ation significantly decreased diaphragm and soleus muscle force production at all stimulation frequencies. Importantly, this observed decrease in force production confirms that azumolene was taken up by the diaphragm and actively blocked the RyR (121) As previously described, 12 hours of MV significantly decreased the specific force produced by the diaphragm compared to Veh (85, 117, 133) T he combination of MV and AZ had an additive effect on MV induced reduction in diaphragm force produ ction.

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85 ROS Production from Permeabilized Diaphragm Fibers It is well established that 12 hours of MV significantly increases ROS emission from diaphragm mitochondria (60, 92) Blockade of the RyR with AZ was not sufficient to prevent this increase (Figure 5 4). These data suggest that the MV induced increase in mitochondrial ROS release is not caused by leakage of calcium through the RyR. M V induced Proteolytic Activity Both calpain and caspase 3 are required proteases for VIDD (85) and increases in the active form of both proteases is well documented following 12 hours of MV (85, 92, 118, 133) Azum olene treatment during MV did not attenuate the MV induced increases in cleaved calpain 1 (Figure 5 5A) or cleaved caspase 3 (Figure 5 5B). Additionally, both calpain and caspase II spectrin. Levels of specific cleavage products can be detected via Western blotting, and provide an index of calpain and caspase 3 activity over the last several hours of the experiment (129) II spectrin are indicative of increased calpain activity, and increases in the 120 kDa fragment indicate caspase 3 activity. Both calpain (Figure 5 5C) and caspase 3 (Figure 5 5D) activity was increased in both the MV and MVA groups compared to their respective controls. Thus, blocking the RyR was not sufficient to prevent the activity of either of these proteases. MV induced Diaphragm Muscle Atrophy Similar to previous reports, 12 hours of MV led to significant decreases in the CSA of Type I, Type IIA, and Type IIB/IIX diaphragm muscle fibers (Figure 5 6A). Azumolene treatment did not attenuate these MV induced decreases in CSA. Representative images of muscle cross sections stained for MHC appear in Figures 5 6B through 5 6E.

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86 Mitochondrial Function Our data agree with previous results demonstrating that 12 hours of MV significantly increases state 4 respiration and significantly decreases RCR (60, 92, 118) (Table 5 3) Interestingly, AZ treatment prevented bo th the MV induced increase in state 4 respiration and the decrease in RCR induced by prolonged MV. To determine if protection of mitochondrial function was associated with preserved mitochondrial ultrastructure, bundles of diaphragm fibers were visualized by electron microscopy. In this regard, mitochondrial structure in animals treated with the vehicle (i.e., Veh) (Figure 5 7A) and AZ animals (Figure 5 7C) appear similar. Diaphragms muscle fibers from animals exposed to MV demonstrate a loss of Z line prot eins, myofilament disorganization, SR swelling, and swollen mitochondria with disrupted cristae (Figure 5 7B). In contrast, MVA diaphragms appear to show some preservation of Z line proteins with myofilaments that appear to be cut, compared to the more dis organized myofilaments found in MV animals (Figure 5 7D). Interestingly, the mitochondria in diaphragm muscles from MVA animals appear more condensed, and disrupted cristae are rare. Additionally, MVA samples do not demonstrate the magnitude of SR swelling observed in MV animals. Autophagy Autophagy is the proteolytic system used to remove damaged mitochondria from skeletal muscle and other cells. Therefore, it is feasible that differences in the rate of autophagy between the experimental groups could expla in the AZ mediated protection of diaphragm mitochondrial function during MV. W e determined the level of autophagic vesicles present in the diaphragm by assessing the ratio of the two forms of lysosomal protein microtubule associated protein light chain 3 ( LC3) During autophagy, LC3 is

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87 conjugated with phosphatidylet hanolamine (PE) (83) This lipidation of LC3 converts LC3 I (without PE) to LC3 II (with PE) This conversion occurs as part of the formation of autophagic vesicles, and a n i ncrease in the ratio of LC3 II/LC3 I is representative of an increas ed presence of these vesicles MV induced a significant increase in LC3 II/I ratio (Figure 5 8), and AZ treatment did not alter the MV induced increase in LC3 II/LCE I. Additionally, si milar numbers of autophagic vesicles were observed in electron micrographs of MV and MVA diaphragm tissue, with observation of vesicles in tissue from MV and MVA animals being much more frequent than in non ventilated animals (data not shown). Thus, altera tions in autophagy ar e an unlikely explanation for AZ mediated protection of the diaphragm mitochondria from MV induced dysfunction. SR Structural Changes following Prolonged MV In addition to the diaphragm mitochondrial structural changes that occur following prolonged MV the electron micrographs clearly demonstrate significant swelling of the SR in diaphragm fibers following 12 hours of MV, and treatment with AZ prevents this swelling. In addition to SR swelling, expression of calsequest rin, the major calcium binding protein of the SR, decreased in the diaphragm following 12 hours of MV (Figure 5 9), and AZ treatment did not prevent this decrease. Taken together, these results suggest that 12 hours of prolonged MV leads to changes in the s tructure and contents of the SR, and that AZ treatment during MV impacts these changes. Discussion Collectively, these experiments do not support the hypothesis that leakage of calcium through the RyR is responsible for increased mitochondrial ROS product ion in the diaphragm following 12 hours of MV. Additionally, treatment of animals with

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88 azumolene is not sufficient to prevent the MV induced activation of calpain or caspase 3 or prevent MV induced atrophy of diaphragm muscle fibers. Interestingly, treatme nt with AZ during prolonged MV is sufficient to prevent MV induced mitochondrial dysfunction in the diaphragm. These findings are discussed in more detail in the subsequent sections. MV induced ROS Emission, Protease Activation, and Atrophy in the Diaphrag m Based on recent evidence, we hypothesized that increased cytosolic calcium, and specifically leakage of calcium through the RyR, is responsible for the MV induced increase in diaphragm mitochondrial ROS production, subsequent protease activation, and fiber atrophy. AZ treatment during prolonged MV was not sufficient to prevent either known cause of MV induced atrophy: increased mitochondrial ROS production (92) or activity of the proteases calpain or caspase 3 (85) Hence, it is not surprising that AZ treatment did not attenuate MV induced diaphragmatic atrophy. Together, our findings imply that leakage of calcium through the RyR is not an important part of the signaling pathway leading to VIDD. Prevention of MV induced Mitochondrial Dysfunction by Azumolene Interestingly, AZ treatment was sufficient to prevent the MV induced mitochondrial dysfunction in the diaphragm that has previously been associated with increased mitochondrial ROS production (60, 92) This protection of mitochondrial respiration was supported by electron micrographs demonstrating that AZ treatment prevented MV induced mitochondrial swelling and cristae disruption. This mitochondrial protection does not appear to have resulted from alterations in autophagy signaling, su ggesting another mechanism must be responsible for this protection. Differences in

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89 calcium uptake by the mitochondria of MVA animals may be responsible for MV induced mitochondrial dysfunction. A potential explanation of this finding follows. As discussed above, small increases in mitochondrial calcium stimulate ATP production (20, 87) but large increases in mitochondrial calcium lead to mitochondrial swelling and dysfunction (20, 137) AZ treatment may have prevented pathological increases in mitochondrial calcium concentrations, thereby preventing mitochondrial swelling and dysfunction. Although no t identified in skeletal muscle, a RyR isoform similar to the skeletal muscle RyR exists in the inner mitochondrial membrane of cardiac mitochondria (11) This RyR functions as a calcium channel, as b oth in vitro calcium induced mitochondrial swelling and calcium uptake is prevented by treatment with dantrolene, a less water soluble analogue of AZ (11, 12) Therefore, our findings could be explained by the presence of a similar RyR in diaphragm mitochondria. Nonetheless, additional research is required to support or deny this postulate. MV induced SR Swelling in Diaphragm F ibers An unexpected finding in these experiments was the AZ prevention of swelling of the SR in diaphragm fibers following 12 hours of MV. While not conclusive, this swelling could be indicative of store operated calcium entry (SOCE) into diaphragm fibers during prolong MV. This concept is supported by the loss of calsequestrin protein during MV, which is associated with SOCE (63, 138) SOCE occurs when th e level of calcium contained within the SR is low and functions to replenish calcium lost due to mitochondrial uptake or to the extracellular space. At least two mechanisms of SOCE exist in cells, with the cause of low SR calcium levels determining which t ype of SOCE occurs (88) One type of SOCE requires a functional RyR receptor, and is inhibited by AZ (139) Although our data are not

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90 conclusive, if SOCE occurred during MV and was inhibited by AZ, it is unlikely that SOCE is responsible for MV indu ced atrophy or protease activation. Experimental Limitations Similar to all investigations, the current expe riments have limitations. First, in our control animals, both the AZ treated and Veh animals were sacrificed immediately following infusion of either AZ or vehicle, instead of maintained under anesthesia for 12 hours prior to sacrifice. The reason for this experimental approach is that during our preliminary experiments, animals stopped breathing when AZ was infused into the jugular vein; hence animals treated with AZ cannot survive for 12 hours under anesthesia without ventilator assistance. Additionally, our AZ animals received only an acute dose of AZ, while the MVA animals received a constant infusion of AZ calculated based on the reported range of dantrolene half lives (e.g., 6 hours). Thus, we are unable to separate the effects of 12 hours of AZ from 1 2 hours of MV. Perhaps the most puzzling result of our experiments is the similar level of calpain activation between MV and MVA animals. Activation of calpain requires increased cytosolic calcium. However, AZ treatment is known to decrease intracellular calcium levels to less than 50% of control levels in resting skeletal muscle (2) One potential explanation of the shrinkage of MVA mitochondria is that, in an effort to increase cytosolic calcium enough to activate calpain, the mitochondria actually exported calcium to increase cytosolic calcium levels. Mitocho ndria contain a sodium calcium exchange pump which could be used for this purpose (20) This also could explain the condensed structure of mitochondria in MVA animals. Additional experiments will be required to determine the level to which cytosolic calcium must rise in vivo to activate calpain and how this level of calcium was achieved in these experiments.

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91 Conclusions In conclusion, calcium leak through the RyR does not appear to be an important signal for increased mitochondrial ROS prod uction during 12 hours of MV. It follows that the activation of calpain in the diaphragm during prolonged MV does not result from RyR leakage. Therefore, further research will be required to identify the signals that lead to both calpain activation and inc reas es mitochondrial ROS production during prolonged MV.

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92 Figure 5 1. Experimental Design to address Aim 3.

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93 Table 5 1. Body w eights. CON MV Vehicle 2696 2697 AZ 27013 27212 Data is presented as meanSD. No significant differences between groups. Table 5 2. Blood gas measurements during MV. MV MVA BP 101 9 101 12 HR 343 20 345 19 CO 2 36 3 37 4 O 2 78 9 70 9 pH 7.43 0.03 7.40 0.02 Ca 2+ 0.98 0.03 0.98 0.05 Data is presented as mean SD. No significant differences between groups.

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94 Figure 5 2 Diaphragm Contractile Function. Specific force was measured in strips of diaphragm. Values are mean s SEM. a: interaction effect; MV depresses force onl y in vehicle treated, AZ depresses force in either pair. b: 2 main effects; MV depresses force in vehicle treated; AZ depresses force in either pair. c: 2 main effects; MV and AZ depress force in both pairs.

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95 Figure 5 3 Soleus Contractile Funct ion. Specific force was measured in an entire soleus. Values are mean s SEM. a: interaction effect; MV depresses force only in vehicle treated, AZ depresses force in either pair. b: 2 main effects; MV and AZ depress force in both pairs. c: 2 main effects; MV depresses force in either pair; AZ depresses force in vehicle treated.

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96 Figure 5 4 Reactive oxygen species emission. Hydrogen peroxide (H2O2) release from permeabilized diaphragm fibers Data were analyzed by two way ANOVA. Values are presented as fold control of mean arbitrary units SEM. *= significantly different from matched control ( p < .05).

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97 A) B) C ) D) Figure 5 5 A ctivity of calpain and caspase 3. A) Levels o f active calpain 1 was detected by Western blot. B) Levels of active caspase 3 was detected by Western blot. C II spectrin cleavage product of calpain (145 kD) was detected via Western blot D II spectrin cleavage product of caspase 3 (120 kD) was detected by Western blot. Values are presented as tubulin = significantly different from matched control ( p < .05)

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98 A) B) C) D) E) Figure 5 6. Cross sectional area and representative images. A) Quantification of diaphragm muscle fiber cross sectional area ( CSA ) by fiber type. Values are mean SEM. *= significantly different from matched control ( p < .05). B E ) Representative staining of MHC I ( blue ), MHC IIa ( green ), and dystrophin ( red ) proteins in a vehicle treated cross section of B) Veh C ) MV D ) AZ E ) MVA.

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99 Table 5 3. Mitochondrial f unction. Veh MV AZ MVA State 3 respiration, nmol O 2 mg min 11.002.14 9.731.37 10.781.32 10.701.37 State 4 respiration, nmol O 2 mg min 1.490.30 2.540.45* 1.630.28 1.900.33 RCR 7.461.13 3.840.47* 6.671.28 5.930.69 Oxygen consumption from permeabilized diaphragm fibers was determined during both State 3 (ADP stimulated) and State 4 (oligomycin inhibited). The respiratory control ratio (RCR) is the ratio of these two values and is a measure of mitochondrial coupling. Data are presented as means SD. No significant differences were found in State 3, but both State 4 and RCR demonstrated interaction effects. *= significantly different from Veh ( p < .05).

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100 A) B) C) D) Figure 5 7. Representative EM pictures of A) Veh B) MV C) AZ D) MVA

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101 Figure 5 8. Ratio of LC3 II/I. As a marker of autophagosome formation, the ratio of LC3 II/I was measured by Western blot. Figure 5 9. Expression of calsequestrin. Protein expression of calsequestrin was measured by tubulin on the same membrane.

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102 CHAPTER 6 DISCUSSION The results of the experiments contained within this dissertation contribute in several important ways to the understanding of the required signals to p romote muscle disuse atrophy. Specifically, these studies co nfirm the role of mitochondria produced reactive oxygen species (ROS) as an important signal that promotes muscle atrophy during prolonged periods of disuse. Our work also demonstrates that calpai n and caspase 3 are key proteases in the development of limb muscle atrophy during prolonged periods of contractile inactivity. Further, our studies indicate that disuse induced calcium release by the RyR is not the mechanism responsible for increased in M V induced increases in reactive oxygen species emission from diaphragm mitochondria. The remainder of this chapter will briefly discuss how these findings can influence future research directions in this field. Results from Chapter 3 clearly demonstrate t hat increased mitochondrial ROS emission is responsible for inactivity induced activation of all four key proteolytic systems in skeletal muscle. Moreover, the work presented in chapter 4 confirms the importance that calpain and caspase 3 play casting indu ced muscle atrophy in rats. Indeed, both of these proteases play a required role in disuse induced limblimb muscle atrophy. Importantly, this study is the first to reveal that a regulatory cross talk exists between calpain and caspase 3 in locomotor skelet al muscle undergoing immobilization induced atrophy. This is an important result and forthcoming experiments should focus on the cellular signaling mechanisms responsible for this regulatory crosstalk in limb muscle.

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103 These experiments are also the first to demonstrate that inhibition of calpain activity protects limb skeletal muscle against disuse induced mitochondrial dysfunction. This exciting finding sets the stage for future experiments to elucidate the mechanism(s) responsible for calpains impact on mi tochondrial function. Indeed, understanding how calpain damages mitochondrial function could identify a biological target for therapeutic intervention. Perhaps the most effective target for interventions to prevent disuse atrophy in skeletal muscle is to determine the mechanisms responsible for inactivity induced increases in mitochondrial ROS production. The results of Chapter 5 suggest that calcium leak through the RyR is not sufficient to induce increases in mitochondrial ROS emission in the diaphragm d uring 12 hours of MV, so other hypotheses will need to be tested. A popular hypothesis is that the diaphragm mitochondria begin to produce more ROS simply by the diaphragm becoming inactive (i.e., shift from state 3 to state 4 respiration), as in vitro stu dies indicate that muscle mitochondria produce more ROS when not actively producing ATP (60) Additional work is required to determine if this phenomenon occurs in vivo and to ascertain if this shift to inactivity increases ROS production sufficiently to account for all of the increased mitochondrial ROS emission observed in the diaphragm during prolonged MV. Additional hypotheses about the root cause of increased ina ctivity induced increases in muscle mitochondrial ROS production also exist. One such hypothesis is that a shift in mitochondrial dynamics occurs during muscle mitochondria during prolonged inactivity, including alterations in the rates of mitochondrial fi ssion and fusion. A recent study by Romanello et al. (101) in mouse hindlimb muscle demonstrated that

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104 genetically inhibiting mitochondrial fission attenuates muscle atrophy due to either fasting or denervation and that overexpression of a specific fission protein alone is sufficient to induce atro phy in skeletal muscle. Additionally, smaller mitochondria, likely due to an increase in mitochondrial fission, appear to produce more ROS (135, 136) Hence, additional experiments that explore the role of mitochondrial dynamics play in disuse muscle atrophy are warranted. In conclusion, much work remains to be done to fully elucidate the signal ing mechanisms that regulate disuse induced skeletal muscle atrophy. A detailed understanding these mechanisms will promote the development of targeted therapies to prevent the problems associated with severe muscle wasting in both limb and respiratory mus cles. Additionally, therapies that protect against disuse atrophy may also be useful against atrophy induced by disease such as cancer and diabetes.

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105 LIST OF REFERENCES 1. Adams GR, Caiozzo VJ, and Baldwin KM Skeletal muscle unweighting : spaceflight and ground based models. J Appl Physiol 95: 2185 2201, 2003. 2. Allen PD, Lpez JR, Snchez V, Ryan JF, and Sreter FA EU 4093 decreases intracellular [Ca2+] in skeletal muscle fibers from control and malignant hyperthermia susceptible swine. Anesthesiology 76: 132 138, 1992. 3. Anderson EJ, and Neufer PD Type II skeletal myofibers possess unique properties that potentiate mitochondrial H(2)O(2) generation. Am J Physiol Cell Physiol 290: C844 851, 2006. 4. Andersson DC, Betzenhauser MJ, Reike n S, Meli AC, Umanskaya A, Xie W, Shiomi T, Zalk R, Lacampagne A, and Marks AR Ryanodine receptor oxidation causes intracellular calcium leak and muscle weakness in aging. Cell Metab 14: 196 207, 2011. 5. Andersson DC, Meli AC, Reiken S, Betzenhauser MJ, Umanskaya A, Shiomi T, D'Armiento J, and Marks AR Leaky ryanodine receptors in beta sarcoglycan deficient mice: a potential common defect in muscular dystrophy. Skelet Muscle 2: 9, 2012. 6. Anzueto A, Peters JI, Tobin MJ, de los Santos R, Seidenfeld JJ, Moore G, Cox WJ, and Coalson JJ Effects of prolonged controlled mechanical ventilation on diaphragmatic function in healthy adult baboons. Crit Care Med 25: 1187 1190, 1997. 7. Appell HJ, Ascenso A, Natsis K, Michael J, and Duarte JA Signs of Necrosis a nd Inflammation Do Not Support the Concept of Apoptosis as the Predominant Mechanism During Early Atrophy in Immobilized Muscle. Basic Appl Myol: 2004, p. 191 196. 8. Arrington DD, Van Vleet TR, and Schnellmann RG Calpain 10: a mitochondrial calpain and i ts role in calcium induced mitochondrial dysfunction. Am J Physiol Cell Physiol 291: C1159 1171, 2006. 9. Bechet D, Tassa A, Taillandier D, Combaret L, and Attaix D Lysosomal proteolysis in skeletal muscle. Int J Biochem Cell Biol 37: 2098 2114, 2005. 10. Betters JL, Criswell DS, Shanely RA, Van Gammeren D, Falk D, Deruisseau KC, Deering M, Yimlamai T, and Powers SK Trolox attenuates mechanical ventilation induced diaphragmatic dysfunction and proteolysis. Am J Respir Crit Care Med 170: 1179 1184, 2004. 1 1. Beutner G, Sharma VK, Giovannucci DR, Yule DI, and Sheu SS Identification of a ryanodine receptor in rat heart mitochondria. J Biol Chem 276: 21482 21488, 2001.

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106 12. Beutner G, Sharma VK, Lin L, Ryu SY, Dirksen RT, and Sheu SS Type 1 ryanodine receptor in cardiac mitochondria: transducer of excitation metabolism coupling. Biochim Biophys Acta 1717: 1 10, 2005. 13. Bialek P, Morris C, Parkington J, St Andre M, Owens J, Yaworsky P, Seeherman H, and Jelinsky SA Distinct protein degradation profiles are in duced by different disuse models of skeletal muscle atrophy. Physiol Genomics 43: 1075 1086, 2011. 14. Blakemore SJ, Rickhuss PK, Watt PW, Rennie MJ, and Hundal HS Effects of limb immobilization on cytochrome c oxidase activity and GLUT4 and GLUT5 protein expression in human skeletal muscle. Clin Sci (Lond) 91: 591 599, 1996. 15. 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, and Gla ss DJ Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294: 1704 1708, 2001. 16. Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ, and Yancopoulos GD Akt /mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol 3: 1014 1019, 2001. 17. Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing th e principle of protein dye binding. Anal Biochem 72: 248 254, 1976. 18. Brand MD Uncoupling to survive? The role of mitochondrial inefficiency in ageing. Exp Gerontol 35: 811 820, 2000. 19. Brocca L, Cannavino J, Coletto L, Biolo G, Sandri M, Bottinelli R and Pellegrino MA The time course of the adaptations of human muscle proteome to bed rest and the underlying mechanisms. J Physiol 590: 5211 5230, 2012. 20. Brookes PS, Yoon Y, Robotham JL, Anders MW, and Sheu SS Calcium, ATP, and ROS: a mitochondrial love hate triangle. Am J Physiol Cell Physiol 287: C817 833, 2004. 21. Caron AZ, Drouin G, Desrosiers J, Trensz F, and Grenier G A novel hindlimb immobilization procedure for studying skeletal muscle atrophy and recovery in mouse. J Appl Physiol 106: 2049 2059, 2009. 22. Chen M, He H, Zhan S, Krajewski S, Reed JC, and Gottlieb RA Bid is cleaved by calpain to an active fragment in vitro and during myocardial ischemia/reperfusion. J Biol Chem 276: 30724 30728, 2001.

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107 23. Clark BC In vivo alterations in skel etal muscle form and function after disuse atrophy. Med Sci Sports Exerc 41: 1869 1875, 2009. 24. Coker RH, and Wolfe RR Bedrest and sarcopenia. Curr Opin Clin Nutr Metab Care 15: 7 11, 2012. 25. DeRuisseau KC, Kavazis AN, Deering MA, Falk DJ, Van Gammere n D, Yimlamai T, Ordway GA, and Powers SK Mechanical ventilation induces alterations of the ubiquitin proteasome pathway in the diaphragm. J Appl Physiol 98: 1314 1321, 2005. 26. Deruisseau KC, Kavazis AN, and Powers SK Selective downregulation of ubiqui tin conjugation cascade mRNA occurs in the senescent rat soleus muscle. Exp Gerontol 40: 526 531, 2005. 27. DeRuisseau KC, Shanely RA, Akunuri N, Hamilton MT, Van Gammeren D, Zergeroglu AM, McKenzie M, and Powers SK Diaphragm unloading via controlled mech anical ventilation alters the gene expression profile. Am J Respir Crit Care Med 172: 1267 1275, 2005. 28. Dodd SL, Gagnon BJ, Senf SM, Hain BA, and Judge AR Ros mediated activation of NF kappaB and Foxo during muscle disuse. Muscle Nerve 41: 110 113, 201 0. 29. Dodd SL, Hain B, Senf SM, and Judge AR Hsp27 inhibits IKKbeta induced NF kappaB activity and skeletal muscle atrophy. FASEB J 23: 3415 3423, 2009. 30. Du J, Wang X, Miereles C, Bailey JL, Debigare R, Zheng B, Price SR, and Mitch WE Activation of caspase 3 is an initial step triggering accelerated muscle proteolysis in catabolic conditions. J Clin Invest 113: 115 123, 2004. 31. Enns DL, Raastad T, Ugelstad I, and Belcastro AN Calpain/calpastatin activities and substrate depletion p atterns during hindlimb unweighting and reweighting in skeletal muscle. Eur J Appl Physiol 100: 445 455, 2007. 32. Esteban A, Anzueto A, Ala I, Gordo F, Apeztegua C, Plizas F, Cide D, Goldwaser R, Soto L, Bugedo G, Rodrigo C, Pimentel J, Raimondi G, and Tobin MJ How is mechanical ventilation employed in the intensive care unit? An international utilization review. Am J Respir Crit Care Med 161: 1450 1458, 2000. 33. Esteban A, Frutos F, Tobin MJ, Ala I, Solsona JF, Valverd I, Fernndez R, de la Cal MA, Benito S, and Toms R A comparison of four methods of weaning patients from mechanical ventilation. Spanish Lung Failure Collaborative Group. N Engl J Med 332: 345 350, 1995. 34. Falk DJ, Deruisseau KC, Van Gammeren DL, Deering MA, Kavazis AN, and Powers SK Mechanical ventilation promotes redox status alterations in the diaphragm. J Appl Physiol 101: 1017 1024, 2006.

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108 35. Falk DJ, Kavazis AN, Whidden MA, Smuder AJ, McClung JM, Hudson MB, and Powers SK Mechanical ventilation induced oxidative stress in th e diaphragm: role of heme oxygenase 1. Chest 139: 816 824, 2011. 36. Fill M, and Copello JA Ryanodine receptor calcium release channels. Physiol Rev 82: 893 922, 2002. 37. Fischer DR, Sun X, Williams AB, Gang G, Pritts TA, James JH, Molloy M, Fischer JE, Paul RJ, and Hasselgren PO Dantrolene reduces serum TNFalpha and corticosterone levels and muscle calcium, calpain gene expression, and protein breakdown in septic rats. Shock 15: 200 207, 2001. 38. Fry CS, Drummond MJ, Lujan HL, Dicarlo SE, and Rasmussen BB Paraplegia increases skeletal muscle autophagy. Muscle Nerve 46: 793 798, 2012. 39. Fuglevand AJ, Bilodeau M, and Enoka RM Short term immobilization has a minimal effect on the strength and fatigability of a human hand muscle. J Appl Physiol 78: 847 855, 1995. 40. Gardiner PF, Favron M, and Corriveau P Histochemical and contractile responses of rat medial gastrocnemius to 2 weeks of complete disuse. Can J Physiol Pharmacol 70: 1075 1081, 1992. 41. Gellerich FN, Gizatullina Z, Nguyen HP, Trumbeckaite S, Vielhaber S, Seppet E, Zierz S, Landwehrmeyer B, Riess O, von Hrsten S, and Striggow F Impaired regulation of brain mitochondria by extramitochondrial Ca2+ in transgenic Huntington disease rats. J Biol Chem 283: 30715 30724, 2008. 42. Gioux M, and Pet it J Effects of immobilizing the cat peroneus longus muscle on the activity of its own spindles. J Appl Physiol 75: 2629 2635, 1993. 43. Goll DE, Thompson VF, Li H, Wei W, and Cong J The calpain system. Physiol Rev 83: 731 801, 2003. 44. Haitsma JJ Diap hragmatic dysfunction in mechanical ventilation. Curr Opin Anaesthesiol 24: 214 218, 2011. 45. Halestrap AP Calcium, mitochondria and reperfusion injury: a pore way to die. Biochem Soc Trans 34: 232 237, 2006. 46. Halestrap AP What is the mitochondrial p ermeability transition pore? J Mol Cell Cardiol 46: 821 831, 2009. 47. Hasselgren PO, and Fischer JE The ubiquitin proteasome pathway: review of a novel intracellular mechanism of muscle protein breakdown during sepsis and other catabolic conditions. Ann Surg 225: 307 316, 1997.

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110 59. Kandarian SC, and Stevenson EJ Molecular events in skeletal muscle during disuse atrophy. Exerc Sport Sci Rev 30: 111 116, 2002. 60. Kavazis AN, Talbert EE, Smuder AJ, Hudson MB, Nelson WB, and Powers SK M echanical ventilation induces diaphragmatic mitochondrial dysfunction and increased oxidant production. Free Radic Biol Med 46: 842 850, 2009. 61. Kelleher AR, Kimball SR, Dennis MD, Schilder RJ, and Jefferson LS The mTORC1 signaling repressors REDD1/2 ar e rapidly induced and activation of p70S6K1 by leucine is defective in skeletal muscle of an immobilized rat hindlimb. Am J Physiol Endocrinol Metab 2012. 62. Knisely AS, Leal SM, and Singer DB Abnormalities of diaphragmatic muscle in neonates with ventil ated lungs. J Pediatr 113: 1074 1077, 1988. 63. Knollmann BC, Chopra N, Hlaing T, Akin B, Yang T, Ettensohn K, Knollmann BE, Horton KD, Weissman NJ, Holinstat I, Zhang W, Roden DM, Jones LR, Franzini Armstrong C, and Pfeifer K Casq2 deletion causes sarcop lasmic reticulum volume increase, premature Ca2+ release, and catecholaminergic polymorphic ventricular tachycardia. J Clin Invest 116: 2510 2520, 2006. 64. Kondo H, Miura M, and Itokawa Y Oxidative stress in skeletal muscle atrophied by immobilization. A cta Physiol Scand 142: 527 528, 1991. 65. Kwak KS, Zhou X, Solomon V, Baracos VE, Davis J, Bannon AW, Boyle WJ, Lacey DL, and Han HQ Regulation of protein catabolism by muscle specific and cytokine inducible ubiquitin ligase E3alpha II during cancer cache xia. Cancer Res 64: 8193 8198, 2004. 66. Lee I, Bender E, and Kadenbach B Control of mitochondrial membrane potential and ROS formation by reversible phosphorylation of cytochrome c oxidase. Mol Cell Biochem 234 235: 63 70, 2002. 67. Lee J, Giordano S, and Zhang J Autophagy, mitochondria and oxidative stress: cross talk and redox signalling. Biochem J 441: 523 540, 2012. 68. Levine S, Nguyen T, Taylor N, Friscia ME, Budak MT, Rothenberg P, Zhu J, Sachdeva R, Sonnad S, Kaiser LR, Rubinstein NA, Powers SK and Shrager JB Rapid disuse atrophy of diaphragm fibers in mechanically ventilated humans. N Engl J Med 358: 1327 1335, 2008. 69. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, and Wang X Cytochrome c and dATP dependent formation of Apaf 1/caspase 9 complex initiates an apoptotic protease cascade. Cell 91: 479 489, 1997.

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111 70. Liu J, Peng Y, Cui Z, Wu Z, Qian A, Shang P, Qu L, Li Y, and Long J Depressed mitochondrial biogenesis and dynamic remodeling in mouse tibialis anterior and gastrocnemius induced by 4 week hindlimb unloading. IUBMB Life 64: 901 910, 2012. 71. Luca M, Smeriglio P, Molinaro M, and Bouch M Unilateral immobilization: a simple model of limb atrophy in mice. Basic Applied Myology: 2008. 72. Maes K, Testelmans D, P owers S, Decramer M, and Gayan Ramirez G Leupeptin inhibits ventilator induced diaphragm dysfunction in rats. Am J Respir Crit Care Med 175: 1134 1138, 2007. 73. Marimuthu K, Murton AJ, and Greenhaff PL Mechanisms regulating muscle mass during disuse atr ophy and rehabilitation in humans. J Appl Physiol 110: 555 560, 2011. 74. 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. 75. McClung JM, Kavazis AN, Whidden MA, DeRuisseau KC, Falk DJ, Criswell DS, and Powers SK Antioxidant admin istration attenuates mechanical ventilation induced rat diaphragm muscle atrophy independent of protein kinase B (PKB Akt) signalling. J Physiol 585: 203 215, 2007. 76. McClung JM, Van Gammeren D, Whidden MA, Falk DJ, Kavazis AN, Hudson MB, Gayan Ramirez G Decramer M, DeRuisseau KC, and Powers SK Apocynin attenuates diaphragm oxidative stress and protease activation during prolonged mechanical ventilation. Crit Care Med 37: 1373 1379, 2009. 77. McClung JM, Whidden MA, Kavazis AN, Falk DJ, Deruisseau KC, a nd Powers SK Redox regulation of diaphragm proteolysis during mechanical ventilation. Am J Physiol Regul Integr Comp Physiol 294: R1608 1617, 2008. 78. Metzger JM, and Fitts RH Contractile and biochemical properties of diaphragm: effects of exercise trai ning and fatigue. J Appl Physiol 60: 1752 1758, 1986. 79. Min K, Smuder AJ, Kwon OS, Kavazis AN, Szeto HH, and Powers SK Mitochondrial targeted antioxidants protect skeletal muscle against immobilization induced muscle atrophy. J Appl Physiol 111: 1459 14 66, 2011. 80. Mitch WE, and Goldberg AL Mechanisms of muscle wasting. The role of the ubiquitin proteasome pathway. N Engl J Med 335: 1897 1905, 1996. 81. Miwa S, and Brand MD Mitochondrial matrix reactive oxygen species production is very sensitive to m ild uncoupling. Biochem Soc Trans 31: 1300 1301, 2003.

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112 82. Mizuno M Human respiratory muscles: fibre morphology and capillary supply. Eur Respir J 4: 587 601, 1991. 83. Mizushima N, and Yoshimori T How to interpret LC3 immunoblotting. Autophagy 3: 542 54 5, 2007. 84. Moylan JS, and Reid MB Oxidative stress, chronic disease, and muscle wasting. Muscle Nerve 35: 411 429, 2007. 85. Nelson WB, Smuder AJ, Hudson MB, Talbert EE, and Powers SK Cross talk between the calpain and caspase 3 proteolytic systems in the diaphragm during prolonged mechanical ventilation. Crit Care Med 2012. 86. O'Leary MF, Vainshtein A, Carter HN, Zhang Y, and Hood DA Denervation induced mitochondrial dysfunction and autophagy in skeletal muscle of apoptosis deficient animals. Am J Ph ysiol Cell Physiol 303: C447 454, 2012. 87. Osellame LD, Blacker TS, and Duchen MR Cellular and molecular mechanisms of mitochondrial function. Best Pract Res Clin Endocrinol Metab 26: 711 723, 2012. 88. Parekh AB, and Putney JW Store operated calcium ch annels. Physiol Rev 85: 757 810, 2005. 89. Picard M, Jung B, Liang F, Azuelos I, Hussain S, Goldberg P, Godin R, Danialou G, Chaturvedi R, Rygiel K, Matecki S, Jaber S, Des Rosiers C, Karpati G, Ferri L, Burelle Y, Turnbull DM, Taivassalo T, and Petrof BJ Mitochondrial Dysfunction and Lipid Accumulation in the Human Diaphragm during Mechanical Ventilation. Am J Respir Crit Care Med 2012. 90. Plant PJ, Bain JR, Correa JE, Woo M, and Batt J Absence of caspase 3 protects against denervation induced skeletal muscle atrophy. J Appl Physiol 107: 224 234, 2009. 91. Powers SK, Demirel HA, Coombes JS, Fletcher L, Calliaud C, Vrabas I, and Prezant D Myosin phenotype and bioenergetic characteristics of rat respiratory muscles. Med Sci Sports Exerc 29: 1573 1579, 199 7. 92. Powers SK, Hudson MB, Nelson WB, Talbert EE, Min K, Szeto HH, Kavazis AN, and Smuder AJ Mitochondria targeted antioxidants protect against mechanical ventilation induced diaphragm weakness. Crit Care Med 39: 1749 1759, 2011. 93. 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.

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113 94. Powers SK, Kavazis AN, and Levine S Prolonged mechanical ventilation alters diaphragmatic structure and f unction. Crit Care Med 37: S347 353, 2009. 95. Powers SK, Kavazis AN, and McClung JM Oxidative stress and disuse muscle atrophy. J Appl Physiol 102: 2389 2397, 2007. 96. Powers SK, Shanely RA, Coombes JS, Koesterer TJ, McKenzie M, Van Gammeren D, Cicale M, and Dodd SL Mechanical ventilation results in progressive contractile dysfunction in the diaphragm. J Appl Physiol 92: 1851 1858, 2002. 97. Powers SK, Smuder AJ, and Criswell DS Mechanistic links between oxidative stress and disuse muscle atrophy. Antioxid Redox Signal 15: 2519 2528, 2011. 98. Powers SK, Smuder AJ, and Judge AR Oxidative stress and disuse muscle atrophy: cause or consequence? Curr Opin Clin Nutr Metab Care 15: 240 245, 2012. 99. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, and Rice Evans C Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26: 1231 1237, 1999. 100. Reed SA, Senf SM, Cornwell EW, Kandarian SC, and Judge AR Inhibition of abolishes skeletal muscle atrophy. Biochem Biophys Res Commun 405: 491 496, 2011. 101. Romanello V, Guadagnin E, Gomes L, Roder I, Sandri C, Petersen Y, Milan G, Masiero E, Del Piccolo P, Foretz M, Scorrano L, Rudolf R, and Sandri M Mitochondrial fission and remodelling contributes to muscle atrophy. EMBO J 29: 1774 1785, 2010. 102. Salazar JJ, Michele DE, and Brooks SV Inhibition of calpain prevents muscle weakness and disruption of sarcomere structure during hindli mb suspension. J Appl Physiol 108: 120 127, 2010. 103. Sandri M Autophagy in skeletal muscle. FEBS Lett 584: 1411 1416, 2010. 104. Sandri M, Lin J, Handschin C, Yang W, Arany ZP, Lecker SH, Goldberg AL, and Spiegelman BM PGC 1alpha protects skeletal musc le from atrophy by suppressing FoxO3 action and atrophy specific gene transcription. Proc Natl Acad Sci U S A 103: 16260 16265, 2006. 105. Sassoon CS Ventilator associated diaphragmatic dysfunction. Am J Respir Crit Care Med 166: 1017 1018, 2002.

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114 106. Sas soon CS, Caiozzo VJ, Manka A, and Sieck GC Altered diaphragm contractile properties with controlled mechanical ventilation. J Appl Physiol 92: 2585 2595, 2002. 107. Sassoon CS, Zhu E, and Caiozzo VJ Assist control mechanical ventilation attenuates ventil ator induced diaphragmatic dysfunction. Am J Respir Crit Care Med 170: 626 632, 2004. 108. Sassoon CS, Zhu E, Fang L, Ramar K, Jiao GY, and Caiozzo VJ Interactive effects of corticosteroid and mechanical ventilation on diaphragm muscle function. Muscle Ne rve 43: 103 111, 2011. 109. Sassoon CS, Zhu E, Pham HT, Nelson RS, Fang L, Baker MJ, and Caiozzo VJ Acute effects of high dose methylprednisolone on diaphragm muscle function. Muscle Nerve 38: 1161 1172, 2008. 110. Schaffer S, and Suleiman M editors. Mito chondria: The Dynamic Organelle New York: Springer, 2007. 111. Senf SM, Dodd SL, and Judge AR FOXO signaling is required for disuse muscle atrophy and is directly regulated by Hsp70. Am J Physiol Cell Physiol 298: C38 45, 2010. 112. Senf SM, Dodd SL, McC lung JM, and Judge AR Hsp70 overexpression inhibits NF kappaB and Foxo3a transcriptional activities and prevents skeletal muscle atrophy. FASEB J 22: 3836 3845, 2008. 113. Shanely RA, Van Gammeren D, Deruisseau KC, Zergeroglu AM, McKenzie MJ, Yarasheski K E, and Powers SK Mechanical ventilation depresses protein synthesis in the rat diaphragm. Am J Respir Crit Care Med 170: 994 999, 2004. 114. Shanely RA, Zergeroglu MA, Lennon SL, Sugiura T, Yimlamai T, Enns D, Belcastro A, and Powers SK Mechanical ventil ation induced diaphragmatic atrophy is associated with oxidative injury and increased proteolytic activity. Am J Respir Crit Care Med 166: 1369 1374, 2002. 115. Slee EA, Keogh SA, and Martin SJ Cleavage of BID during cytotoxic drug and UV radiation induce d apoptosis occurs downstream of the point of Bcl 2 action and is catalysed by caspase 3: a potential feedback loop for amplification of apoptosis associated mitochondrial cytochrome c release. Cell Death Differ 7: 556 565, 2000. 116. Smuder A, Kavazis A, Hudson M, Nelson W, and Powers S Oxidation enhances myofibrillar protein degradation via calpain and caspase 3. Free Radic Biol Med 49: 1152 1160, 2010.

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115 117. Smuder AJ, Hudson MB, Nelson WB, Kavazis AN, and Powers SK Nuclear factor es to mechanical ventilation induced diaphragm weakness*. Crit Care Med 40: 927 934, 2012. 118. Smuder AJ, Min K, Hudson MB, Kavazis AN, Kwon OS, Nelson WB, and Powers SK Endurance exercise attenuates ventilator induced diaphragm dysfunction. J Appl Physi ol 112: 501 510, 2012. 119. Solomon V, and Goldberg AL Importance of the ATP ubiquitin proteasome pathway in the degradation of soluble and myofibrillar proteins in rabbit muscle extracts. J Biol Chem 271: 26690 26697, 1996. 120. Stevens Lapsley JE, Ye F, Liu M, Borst SE, Conover C, Yarasheski KE, Walter GA, Sweeney HL, and Vandenborne K Impact of viral mediated IGF I gene transfer on skeletal muscle following cast immobilization. Am J Physiol Endocrinol Metab 299: E730 740, 2010. 121. Sudo RT, Carmo PL, Trachez MM, and Zapata Sudo G Effects of azumolene on normal and malignant hyperthermia susceptible skeletal muscle. Basic Clin Pharmacol Toxicol 102: 308 316, 2008. 122. Taillandier D, Combaret L, Pouch MN, Samuels SE, Bchet D, and Attaix D The role of ubiquitin proteasome dependent proteolysis in the remodelling of skeletal muscle. Proc Nutr Soc 63: 357 361, 2004. 123. Talbert EE, Smuder AJ, Min K, Kwon OS, and Powers SK Calpain and capase 3 play required roles in immobilization induced limb muscle at rophy. J Appl Physiol 2013. 124. Thomason DB, Biggs RB, and Booth FW Protein metabolism and beta myosin heavy chain mRNA in unweighted soleus muscle. Am J Physiol 257: R300 305, 1989. 125. Tidball JG, and Spencer MJ Expression of a calpastatin transgene slows muscle wasting and obviates changes in myosin isoform expression during murine muscle disuse. J Physiol 545: 819 828, 2002. 126. Torpy JM, Campbell AD, and Glass RM JAMA patient page. Mechanical ventilation. J AMA 303: 902, 2010. 127. Urso ML, Clarkson PM, and Price TB Immobilization effects in young and older adults. Eur J Appl Physiol 96: 564 571, 2006. 128. Van Gammeren 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.

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118 BIOGRAPHICAL SKETCH Erin Talbert is from Russiaville, Indiana. She graduated with a Bachelor of Science degree in chemistry with an emphasis in biochemistry from Purdue University in 2007. She began her doct oral work in the same year at the University of Florida with Dr. Scott Powers. Erin completed her PhD in exercise physiology in 2013, with her work investigating the mechanisms of disuse atrophy of skeletal muscle.