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Biphasic Effects of Nitric Oxide on Skeletal Muscle Myotube Atrophy

Permanent Link: http://ufdc.ufl.edu/UFE0022596/00001

Material Information

Title: Biphasic Effects of Nitric Oxide on Skeletal Muscle Myotube Atrophy
Physical Description: 1 online resource (139 p.)
Language: english
Creator: Soltow, Quinlyn
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: atrophy, b, factor, in, kappa, nitric, nos, nuclear, oxide, stretch, vitro
Health and Human Performance -- 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: Skeletal muscle disuse atrophy occurs during prolonged periods of reduced muscle activity often seen with bed rest, limb immobilization, and space flight. Muscle injury or lack of mechanical activity causes disruptive nitric oxide synthase (NOS) activity, which is sufficient to induce forkhead box O-3a, muscle RING finger-1, and muscle atrophy F-box, and nuclear factor-kappa B (NF-kappaB) through classical and alternative pathways, respectively. Paradoxically, increased nitric oxide production is caused by muscle loading and is essential for muscle growth. This is the first study to develop two completely intrinsic models of skeletal muscle atrophy in vitro: 1) withdrawal from moderate cyclic stretch, and 2) high magnitude cyclic strain. First, moderate cyclic mechanical stretch can be used as a model of activity in cultured skeletal muscle myotubes, and increased myotube size through NOS-dependent Akt signaling. Cessation of moderate stretch caused protein degradation, altered neuronal NOS localization, and a reduction in myotube size via downregulation of Akt, which may contribute to NF-kappaB signaling through an alternative pathway. Secondly, high magnitude cyclic strain induced the classical pathway of NF-kappaB signaling and upregulated inducible NOS. These data demonstrated in vitro models of atrophy independent of external factors and provide evidence to better understand the signaling pathways involved during skeletal muscle loss.
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 Quinlyn Soltow.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Criswell, David S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022596:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022596/00001

Material Information

Title: Biphasic Effects of Nitric Oxide on Skeletal Muscle Myotube Atrophy
Physical Description: 1 online resource (139 p.)
Language: english
Creator: Soltow, Quinlyn
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: atrophy, b, factor, in, kappa, nitric, nos, nuclear, oxide, stretch, vitro
Health and Human Performance -- 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: Skeletal muscle disuse atrophy occurs during prolonged periods of reduced muscle activity often seen with bed rest, limb immobilization, and space flight. Muscle injury or lack of mechanical activity causes disruptive nitric oxide synthase (NOS) activity, which is sufficient to induce forkhead box O-3a, muscle RING finger-1, and muscle atrophy F-box, and nuclear factor-kappa B (NF-kappaB) through classical and alternative pathways, respectively. Paradoxically, increased nitric oxide production is caused by muscle loading and is essential for muscle growth. This is the first study to develop two completely intrinsic models of skeletal muscle atrophy in vitro: 1) withdrawal from moderate cyclic stretch, and 2) high magnitude cyclic strain. First, moderate cyclic mechanical stretch can be used as a model of activity in cultured skeletal muscle myotubes, and increased myotube size through NOS-dependent Akt signaling. Cessation of moderate stretch caused protein degradation, altered neuronal NOS localization, and a reduction in myotube size via downregulation of Akt, which may contribute to NF-kappaB signaling through an alternative pathway. Secondly, high magnitude cyclic strain induced the classical pathway of NF-kappaB signaling and upregulated inducible NOS. These data demonstrated in vitro models of atrophy independent of external factors and provide evidence to better understand the signaling pathways involved during skeletal muscle loss.
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 Quinlyn Soltow.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Criswell, David S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022596:00001


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1 BIPHASIC EFFECTS OF NITRIC OXIDE ON SKELETAL MUSCLE MYOTUBE ATROPHY By QUINLYN ANN SOLTOW A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Quinlyn Ann Soltow

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3 To my family, who always believes in me, even when I do not

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4 ACKNOWLEDGMENTS First, I thank Dr. Criswell for all of his gui dance, kindness, and support during m y growth as a graduate student in his la b. I also thank the rest of my committee members, Drs. Scott Powers, Stephen Dodd, and Glenn Walter, for their numerous suggestions and intellectual discussions. The dedication and passion demonstrat ed in their work has had great influence on my professional career. Second, I would like to acknowledg e all of the current and past members of the Molecular Physiology lab, Jeff Sellman, Jodi Long, Jenna Betters, and Vitor Lira, for teaching me everything that I know about biological assays and cell culture; as well as Jason Drenning and Liz Zeanah for their compassion and friendship both in and out of the lab. I would especially like to thank Vitor for his words of encouragement and advice during our final semester together. The memories of our struggles and laughs will never be forgotten. Finally, I commend all of my family and cl ose friends for putting up with me throughout this entire process. I could not have had the c onfidence and perseverance to obtain this degree had it not been for their endless love and support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF FIGURES .........................................................................................................................7LIST OF ABBREVIATIONS ........................................................................................................ 10ABSTRACT ...................................................................................................................... .............11 CHAP TER 1 INTRODUCTION .................................................................................................................. 12Background .................................................................................................................... .........12Specific Aims and Hypotheses ............................................................................................... 13Clinical Significance ...............................................................................................................14Strengths and Limitations ..................................................................................................... ..152 LITERATURE REVIEW .......................................................................................................18Overview of Disuse Sk eletal Muscle Atrophy ....................................................................... 18Redox Balance ........................................................................................................................20Mechanotransduction ..............................................................................................................21Overview of Akt Pathway Contribution to Atrophy ...............................................................22Disruption of DGC during Unloading .................................................................................... 23Nitric Oxide Contribution to Atrophy .................................................................................... 24Nuclear Factor of B Pathway of Atrophy .............................................................................28Summary ....................................................................................................................... ..........303 MATERIALS AND METHODS ...........................................................................................37Experimental Designs .......................................................................................................... ...37General Methods .....................................................................................................................40Myogenic Culture ............................................................................................................40Primary Culture ............................................................................................................... 41Mechanical Stimulation Using Cyclic Strain ..................................................................41Immunohistochemistry .................................................................................................... 42Image Analysis ................................................................................................................ 43Nuclear and Cytosolic Fractionation ............................................................................... 43Whole Cell Lysate ........................................................................................................... 43Western Blot Analysis .....................................................................................................43Isolation of RNA and Real-Time RT PCR ......................................................................44Nuclear Factor of B-Dependent Transcri ptional Activity .............................................45Dual Luciferase Assay .....................................................................................................45

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6 Nitric Oxide Production .................................................................................................. 46Statistical Analysis .......................................................................................................... ........464 RESULTS ....................................................................................................................... ........53Skeletal Muscle My otube Atrophy Model ............................................................................. 53Myotube Atrophy ............................................................................................................54Protein Degradation .........................................................................................................54Cellular Signaling during Disuse ............................................................................................56Measurements in C2C12 Cell Culture during Disuse .......................................................56Myosin heavy chain and dystrophin ......................................................................... 56Nitric oxide synthase isoforms ................................................................................. 57Localization of nNOS ............................................................................................... 57Nitric oxide production ............................................................................................ 58Components of the Akt pathway .............................................................................. 58Alternative NFB pathway .....................................................................................60Nuclear factor of -B transcriptional ac tivity during disuse ....................................62Neuronal NOS Knockout Primar y Satellite Cell Culture ................................................63Cellular Signaling during High Strain ....................................................................................63Measurements in C2C12 Cell Culture duri ng High Strain ................................................ 64Classical NFB pathway .........................................................................................64Inducible NOS gene expression ...............................................................................66Nuclear factor of -B transcriptional activ ity during high strain .............................66Inducible NOS Knockout Primary Satellite Cell Culture ................................................675 DISCUSSION .................................................................................................................... ...102Main Findings .......................................................................................................................102Disuse Atrophy Model ..........................................................................................................102Cessation of Cyclic Stretch Causes Skeletal Muscle Myotube Atrophy .......................102Localization and Activity of NOS after Stretch ............................................................ 105Stretch-Induced Activation of Akt is NOS-Dependent ................................................. 108Moderate Stretch and Pathways of NFB Signaling ....................................................110Inflammation-Associated Atrophy Model ............................................................................ 113High Magnitude Stretch Induces iNOS and NFB ...................................................... 113Nitric Oxide Synthase Inhibition Prevents NFB Nuclear Translocation, iNOS and MAFbx Expression during High Strain .....................................................................116Inducible NOS Is Not Solely Responsible for NO Production during High Strain ...... 116Limitations and Future Directions ........................................................................................ 117Conclusions ...........................................................................................................................117LIST OF REFERENCES .............................................................................................................121BIOGRAPHICAL SKETCH .......................................................................................................139

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7 LIST OF FIGURES Figure page 1-1 Proposed model of nitric oxide-dependent control of skeletal m uscle size with alterations in loading stimuli. .............................................................................................172-1 Contributions to skeletal musc le protein loss during unloading. ....................................... 312-2 Model of the dystrophin-g lycoprotein complex as a transsarcolemmal linker between the subsarcolemmal cytoskeleton and the extracellular matrix. ........................................322-3 Overview of PI3K/Akt signaling network .........................................................................332-4 Subcellular compartmentalisation of NOS isoforms in skeletal muscle ............................ 342-5 Degree of skeletal muscle atrophy as a function of NO pr oduction during various conditions. ................................................................................................................... .......352-6 Pathways of NFB signaling in cachexia/cytoki ne-induced muscle atrophy (classical pathway) versus disuse muscle atrophy. ............................................................ 363-1 Experiment 1 design for Aims 1 and 2 .............................................................................. 473-2 Experiment 2a and 2b designs for Aim 2. .......................................................................... 483-3 Average 4-amino-5-methylamino-2',7'difluorofluorescein (DAF-FM) diacetate fluorescence in C2C12 myotubes after 1 h of cyclic stretch at various magnitudes ........... 493-4 Experiment 3 design for Aim 2.. ........................................................................................ 503-5 Experiment 4 design for Aim 3.. ........................................................................................ 513-6 Experiment 5 design for Aim 3 .......................................................................................... 524-1 Representative images of C2C12 myotubes using the atrophy protocol. ............................694-2 Image analysis of C2C12 myotubes using the atrophy protocol .........................................714-3 AlphaII-spectrin protein degradation of C2C12 myotubes using the atrophy protocol ....... 724-4 Talin protein degradation of C2C12 myotubes using the atrophy protocol .........................734-5 Integrin 1 protein expression in C2C12 myotubes using the atrophy protocol ................. 744-6 Total protein content of C2C12 myotubes using the atrophy protocol ................................ 754-7 Representative images of C2C12 myotubes immunostained for dystrophin and MHC type IIa and corresponding negative controls after 6 d of differentiation .......................... 76

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8 4-8 Representative images of C2C12 myotubes immunostained for nNOS, iNOS, and eNOS after 6 d of differentiation. ......................................................................................774-9 Representative images of C2C12 myotubes immunostained for nNOS using the atrophy protocol. ............................................................................................................. ...784-10 Total nitrate plus nitr ite concentrations throughout the atrophy protocol .........................804-11 Ratio of phosphorylated to to tal Akt protein expression in C2C12 myotubes 12, 24, and 48 h after 12% stretch .................................................................................................814-12 Ratio of phosphorylated to total GSK-3 protein expression in C2C12 myotubes 12, 24, and 48 h after 12% stretch ...........................................................................................824-13 Phosphorylated FOXO3a protein levels in C2C12 myotubes 12, 24, and 48 h after 12% stretch.........................................................................................................................834-14 Muscle atrophy F-box (MAFbx) protein expression in C2C12 myotubes 12, 24, and 48 h after 12% stretch ........................................................................................................844-15 Nuclear p50 protein levels in C2C12 myotubes 12, 24, and 48 h after 12% stretch ...........854-16 Nuclear Bcl-3 prot ein expression in C2C12 myotubes 12, 24, and 48 h after 12% stretch ....................................................................................................................... ..........864-17 Nuclear and cytosolic p65 protein levels in C2C12 myotubes 12, 24, and 48 h after 12% stretch.........................................................................................................................874-18 Cytosolic I Bprotein expression in C2C12 myotubes 12, 24, and 48 h after 12% stretch ....................................................................................................................... ..........894-19 Nuclear factor of B (NFB) transcriptional activity in C2C12 myotubes 12, 24, and 48 h after 12% stretch ........................................................................................................904-20 Nuclear p50 protein expression in C2C12 myotubes subjected to high cyclic strain for 3 h.......................................................................................................................................914-21 Nuclear and cytosolic p6 5 protein expression in C2C12 myotubes subjected to high cyclic strain for 3 h ......................................................................................................... ...924-22 Cytosolic I Bprotein expression in C2C12 myotubes subjected to high cyclic strain for 3 h .................................................................................................................................944-23 Muscle atrophy F-box (MAFbx) protein expression in C2C12 myotubes subjected to high cyclic strain for 3 h ....................................................................................................954-24 Inducible NOS (iNOS) mRNA levels in C2C12 myotubes during 4 h of high cyclic strain ........................................................................................................................ ...........96

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9 4-25 Nuclear factor of B (NFB) tran scriptional activity in C2C12 myotubes subjected to high cyclic strain for 3 h ....................................................................................................974-26 Nuclear p50 protein expression in primary cultured myotubes from iNOS-/and wildtype mice subjected to high cyclic strain for 3 h ........................................................ 984-27 Nuclear and cytosolic p65 protein expression in primary cultured myotubes from iNOS-/and wildtype mice subjected to high cyclic strain for 3 h .....................................994-28 Cytosolic I Bprotein expression in primar y cultured myotubes from iNOS-/and wildtype mice subjected to high cyclic strain for 3 h. ..................................................... 1015-1 Proposed mechanism of nNOS contribu tion to skeletal muscle disuse atrophy signaling ..................................................................................................................... ......1195-2 Proposed mechanism of NOS involveme nt in inflammation-associated atrophy during high strain ............................................................................................................ .120

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10 LIST OF ABBREVIATIONS Bcl-3 B-cell lymphoma-3 cGMP cyclic guanosine monophosphate DGC dystrophin-glycoprotein complex eNOS endothelial nitr ic oxide synthase FOXO forkhead box O GSK-3 glycogen synthase kinase-3beta I BI kappa B-alpha iNOS inducible nitric oxide synthase L-NAME NG-nitro-L-arginine methyl ester MAFbx muscle atrophy F-box MuRF1 muscle RING finger-1 NFB nuclear factor-kappa B nNOS neuronal nitric oxide synthase NO nitric oxide NOS nitric oxide synthase PI3K phosphoinositide-3-kinase

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy BIPHASIC EFFECTS OF NITRIC OXIDE ON SKELETAL MUSCLE MYOTUBE ATROPHY By Quinlyn Ann Soltow December 2008 Chair: David S. Criswell Major: Health and Human Performance Skeletal muscle disuse atrophy occurs during prolonged periods of reduced muscle activity often seen with bed rest, limb immobilization, and space flight. Muscle injury or lack of mechanical activity causes disruptive nitric oxide synthase (NOS) activity, which is sufficient to induce forkhead box O-3a, muscle RING finger-1, and muscle atrophy F-box, and nuclear factorkappa B (NF-kappaB) through cla ssical and alternative pathways respectively. Paradoxically, increased nitric oxide production is caused by musc le loading and is essential for muscle growth. This is the first study to develop two completely intrinsic models of skeletal muscle atrophy in vitro : 1) withdrawal from moderate cyclic stretch, and 2) high ma gnitude cyclic strain. First, moderate cyclic mechanical stretch can be used as a model of activity in cultured skeletal muscle myotubes, and increased myotube size thr ough NOS-dependent Akt signaling. Cessation of moderate stretch caused protein degradation, altered neuronal NOS localization, and a reduction in myotube size via downregulation of Akt, which may contribute to NF-kappaB signaling through an alternative pathway. Secondly, high magn itude cyclic strain induced the classical pathway of NF-kappaB signaling and upregulated inducible NOS These data demonstrated in vitro models of atrophy independent of external factors and provide evidence to better understand the signaling pathways invol ved during skeletal muscle loss.

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12 CHAPTER 1 INTRODUCTION Background Skeletal m uscle atrophy is a c linically significant problem as it arises from a variety of factors, such as prolonged bed rest, limb im mobilization, space flight, denervation, cancer, sepsis, and ageing. In addition to compromised mobility and balance, loss of muscle mass contributes to the metabolic syndrome and increased risk for heart disease. Mechanical injury or lack of mechanical activity during disuse causes high NOS activity (140, 149, 169), which is sufficient to induce FOXO3a, MuRF1, MAFbx, and skeletal muscle atrophy (11, 169). Paradoxically, increased NO producti on, albeit a lower level than shown in atrophy models, is caused by muscle loading (passive stretch, electrical s timulation, reloading) and is essential for muscle growth (145, 162, 178). Low concen trations of NO can inhibit GSK-3 activity via a cGMP/Akt-dependent mechanism (48) and, therefore, may coordinate FOXO and NFB regulation of atrophy signaling pathways (Figure 1-1). Determining if NO can prevent and/or contribut e to skeletal muscle atrophy by regulating FOXO and NFB is important and may lead to potential therapeutic strategies to alleviate skeletal muscle disuse atrophy. Therefore, we hypothesize that NOS is a vital signal transducer in skeletal muscle, in that mechanical lo ad imposed upon the muscle membrane maintains moderate NO concentrations to preserve skelet al muscle mass. However, cessation of activity causes nNOS to dissociate from the sarcol emma and produce unre gulated amounts of NO contributing to proteolysis and skeletal muscle atrophy. Likewise, excessive strain to the muscle leads to upregulation of iNOS and ac tivation of atrophy signaling pathways.

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13 Specific Aims and Hypotheses Specific aim 1: To develop an in vitro m odel of skeletal muscle disuse atrophy. Cyclic stretch provides strain to th e sarcolemma and through mecha notransduction, increases muscle integrity (213), metabolism (86, 155), NO producti on (178, 212), and protein synthesis (81). We tested if removal of the stretc h stimulus reverses these proces ses to induce protein degradation and atrophy. Hypothesis 1: Cessation of repetitive bouts of cyc lic stretch to myotubes causes protein degradation and a reduction in m yotube size and protein content. Specific aim 2: To test if cessation of repetitive daily activity of skeletal muscle myotubes causes elevation of NO production (>20%) and activa tion of skeletal muscle disuse atrophy. We have shown that moderate magnitudes of cyclic stretch stimulate an approximately 10-20% increase in NO production. Low con centrations of NO inhibit GSK-3 activity, which is hypothesized to be the primary kinase responsible for activation of the NFB complexes (p502/Bcl-3) implicated in disuse atrophy (83, 84). Conversely, higher amounts of NO induce GSK-3 Bcl-3 and FOXO3a (169) activity responsible for atrophy. Hypothesis 2A: Moderate magnitudes of cyclic stretch in C2C12 myotubes maintain nNOS localization, low levels of NO, and oppose skeletal muscle atrophy by inhibition of GSK-3 FOXO3a, and NFB. Hypothesis 2B: Cessation of stretch in C2C12 myotubes causes dissociation of nNOS from the sarcolemma, elevated NO production and initi ation of skeletal muscle disuse atrophy signaling.

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14 Hypothesis 2C: NOS inhibition wi th L-NAME in C2C12 or genetic knockout of nNOS in primary myotubes prevents the upregulation of atr ophy signaling pathways following cessation of cyclic stretch. Specific aim 3: To ascertain if excessive load applie d to skeletal muscle myotubes causes production of high amounts of NO via iNOS and activation of proteoly sis. Skeletal muscle injury leads to inflammation and upregulation of i NOS (140, 147), which is associated with the classical pathway of NFB (I B/p50/p65) (102) to induce pr otein degradation (11). Hypothesis 3A: High tensile strain of C2C12 myotubes induce iNOS, elevated NO production and proteolysis via th e classical pathway of NFB signaling. Hypothesis 3B: NOS inhibition wi th L-NAME in C2C12 or genetic knockout of iNOS in primary myotubes protects myotubes from degr adation following high magnitudes of stretch through inhibition of NFB. Clinical Significance Skeletal m uscle tissue constitutes about 40% of human body mass to maintain basic functions such as locomotion, respiration, and meta bolism. Skeletal muscle has the innate ability to adapt to stessors. Resistance exercise and suffi cient nutrient uptake leads to skeletal muscle hypertrophy characterized by an increase in fiber cross-sectional area, force production, and protein content. On the othe r hand, skeletal muscle atrophy can ensue following periods of inactivity, lack of nutrition, or disease. Atrophy is described as a loss in fiber size, protein content, and strength. Skeletal muscle atrophy is a product of and can contribute to numerous disease states. A loss of skeletal muscle leads to fatigability, a loss in mobility and insulin resistance; all of which are risk factors for type II diabetes and cardiovascular disease.

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15 Much work has been done in the field of skeletal muscle at rophy. Various models, including limb immobilization, limb unloading, denervation, and endotoxin administration, have been used to research the causes of musc le atrophy. These models are all performed in vivo and can provide little insight on the intrinsic factors le ading to muscle degradation in the absence of humoral and neural influence. Therefore, an in vitro model of skeletal muscle atrophy, applying both disuse and inflammation-asso ciated types, is necessary to study the signaling pathways leading to loss of muscle mass. El ucidating these signals may lead to therapeutic strategies, such as pharmaceutical interventions or rehabilitation t echniques, to attenuate or eliminate skeletal muscle atrophy as a cause or side effect of disease. Strengths and Limitations W ith this project, we developed a novel approa ch to the study of sk eletal muscle disuse atrophy. Although informative research can be done using in vivo models of atrophy (hindlimb suspension, immobilization, denervat ion), there is a need for an in vitro model that mimics the same signaling pathways revealed during the in vivo studies independent of potential extraneous effects of neural and humoral factors. The few in vitro models of atrophy that have been investigated include starvati on, glucocorticoid treatment, and cytokine administration (interleukins and tumor necrosis factor-alpha (TNF) (15, 126, 168). Although these strategies induce atrophy of myotubes, they do so by act ing through the TNF recep tor and the classical pathway of NFB signaling (I B/p50/p65). New research sugge sts this pathway does not account for inactivity-related atr ophy (83, 84); therefore, an in vitro model has yet to be designed which activates the alternative pathway of NFB signaling (p50/Bcl-3). Th erefore, this is the first in vitro model of disuse atrophy to act via the same mechanisms seen in vivo

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16 By using cessation of stretch to induce di suse signaling and high strain to cause inflammation associated-atrophy, we are employi ng a completely intrinsic model which is independent of any external physiological influence. One limitation inherent to in vitro models, such as this, is the need to confirm observations in vivo Nevertheless, this study establishes intrinsic cellular mechanisms and will lead to in vivo studies to confirm the physiological significance of NO in skeletal muscle disuse atrophy.

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17 Figure 1-1. Proposed model of n itric oxide-dependent control of skeletal muscle size with alterations in loading stimuli.

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18 CHAPTER 2 LITERATURE REVIEW Skeletal m uscle atrophy is a c linically significant problem as it arises from a variety of factors, such as prolonged bed rest, limb im mobilization, space flight, denervation, cancer, sepsis, and ageing. Loss of muscle mass has been shown to contribute to inactivity, obesity, insulin resistance, hypertension, and hyperlipidem ia (altogether termed metabolic syndrome). Minimization of muscle atrophy and its side eff ects will prevent the progression to more serious pathological disorders such as cardiovascular disease and cancer and improve overall quality of life. Overview of Disuse Skeletal Muscle Atrophy Although skeletal m uscle atrophy occurs with nu merous pathologies such as cancer, sepsis, and diabetes (73, 87), muscle atrophy can also occur in the absence of disease during prolonged periods of reduced muscle activity (20). It is well established that prolonged best rest, limb immobilization, mechanical ventilation, or space flight can produce muscle atrophy in humans. However, investigation of mechanisms responsib le for disuse atrophy in humans is difficult. Therefore, animal models have been utilized to mimic the various conditions that produce human disuse atrophy. For example, to imitate prolonged bed rest and space flight in humans, animal models of hindlimb suspension are used to unload the hindlimb locomotor muscles. Also, animal models of limb immobilization are commonly used to research disuse atrophy. According to research employing both the af orementioned models, disuse muscle atrophy occurs due to both a decrease in muscle protei n synthesis and an increase in the rate of proteolysis (21, 176). After the onset of muscle unloading, the rate of protein synthesis declines rapidly and reaches a new steady-state level at ~ 48 hours (176). The drop in protein synthesis is followed by a large and rapid increase in protein degradation peaking at approximately 14 days

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19 (Figure 2-1). Fiber type disparities also affect the extent of musc le atrophy. Solei (predominantly type I fibers) show the greatest degree of atrophy during unloading and immobilized conditions (20). Collectively, the reduced activity of skeletal muscle negatively impacts muscle mass through alterations of the rates of protein synt hesis and degradation th at ultimately lead to muscle atrophy. Although a decrease in protein s ynthesis initiates th e drop in muscle mass, the predominant cause of atrophy is the dramatic increase in protei n degradation. Therefore, strategies to attenuate proteolysis will be the most beneficial to c ounteracting muscle atrophy. Several proteolytic systems contribute to the degradation of musc le proteins. They include lysosomal proteases, calcium-activated proteases, and the proteasome system. Lysosomal proteases are activated in skeletal muscle undergoing disuse atrophy; however, the contribution of these proteases appears limited (58, 74, 139). It appears the bulk of muscle proteolysis involves both calpain and the proteasome system (139), and to some lesser exte nt, caspase-3 (50). The proteasome system can degrade monomeric contractile proteins, however it is unable to degr ade intact actomyosin complexes (65). Therefore, myofilaments must be released from the sarcomere before degradation by the proteasome system can occu r. Evidence indicates that both calpain and caspase-3 are capable of producing actomyosin disa ssociation which is believed to be the ratelimiting step in protein degradation. The proteasome system consists of the 20S core proteasome and the 26S proteasome complex (20S core + 19S regulatory complex) (43). The 26S proteasome recognizes ubiquitinlabeled proteins, unfolds, and degrades these pr oteins via an ATP-dependent mechanism. The binding of ubiquitin to protein substrates requires the ubiqu itin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitinligase enzymes (E3). Specific E3 ligases

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20 termed MAFbx and MuRF1 have been discovered in skeletal muscle and are essential in almost all models of skeletal muscle atrophy (19, 67). Redox Balance A potential m echanism that would trigger in creased protein degrad ation and atrophy in skeletal muscle is oxidative stress, where an tioxidant protein and scavenger protection are overwhelmed by oxidant production. Oxidation can al ter the structure and function of proteins, lipids, and nucleic acids, leading to cellular injury and even cell death. Kondo and colleagues provided the first eviden ce that oxidants contributed to disuse muscle atrophy. This work revealed that immobiliz ation of skeletal muscles was associated with oxidative injury in the muscle and the oxidant injury can co ntribute to muscle atrophy (101). Oxidative stress also increases in hindlimb mu scle (soleus) with hindlimb unloading. Hindlimb unloading is linked to an imbalance in the an tioxidant system and in creased hydroperoxides. Unloading-induced disruption of the antioxidant enzyme and scavenger profile could also predispose skeletal muscle to inflammation and muscle damage upon reloading (108). The exact mechanisms of how oxidative stre ss induces proteolysis are still unknown, but many possibilities exist. First, oxidative stress decreases membrane calcium-ATPase activity thereby retarding calcium removal from the cell and contributing to cellular calcium accumulation (160). Increased intracellular calciu m levels would activate calpain and caspase resulting in augmented proteolysis. Second, oxi dative stress upregulat es the expression of MAFbx and MuRF1 and augments the 26S prot easome which would lead to accelerated proteolysis and muscle atrophy (19, 112). At present is uncer tain which oxidant-producing pathways are responsible for the oxidative injury w ithin inactive skeletal muscles, and oxidative stress in skeletal muscle may be due to the in teraction of different ox idant production pathways.

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21 Increased oxidant production during unloading may be due to a lack of neural stimulation or to reduced mechanical perturbation on the sarcolemma. Re duced nerve input can disrupt calcium release, reuptake and signaling within the sarcoplasm and stimulate calcium-activated proteolytic pathways. A decrement in contractil e activity and metabolic demand may also disturb oxidative phosphorylation in the mitochondria unbalancing the redox state. Finally, and the focus of this proposal, is the lack of mechanical perturbation of th e sarcolemma. Redu ced mechanical strain on the muscle membrane may impair signaling from force transducers in the extracellular matrix to the cytoskeleton. The prolonged reduc tion in tension on the mu scle fiber can cause dissociation of these mechanotransduction prot eins and activate atroph ic signaling pathways. Nitric oxide synthase is one such protein that can become dissociated from a complex of molecules localized at the membrane of muscle and produce oxidant stress during periods of unloading (169). The nitric oxide synthase pathway has been impli cated in disuse atrophy and will be discussed in more detail. Mechanotransduction The dystrophin-glycoprotein com plex (DGC ) provides a transmembrane association between the extracelluar matrix and various in tracellular proteins and protein complexes, comparable to other transmembrane complexes (Figure 2-2), such as the integrins (55). Disruption of the DGC, such as occurs in Du chenes muscular dystrophy or the mdx mouse, results in repetitive skeletal muscle injury/r egeneration and muscle atrophy. Focal adhesionassociated proteins, including vi nculin, talin, paxillin, and focal adhesion kinase (FAK), are associated with the DGC. Talin and vinculin are functionally similar to dystrophin because they serve as links between the actin cytoskeleton and a transmembrane recep tor for extracellular matrix molecules (11, 102), and th ey are involved in the transmi ssion of force between the actin cytoskeleton and the cell membrane. Integrin, the transmembrane binding partner for talin and

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22 vinculin, is expressed at higher levels in mdx muscles (73), and recent investigations have shown that the transgenic overexpression of 7-integrin in mdx musc les could greatly reduce dystrophinopathy. However, the integrin complex is also rich in signalin g molecules, so the possibility remains that repair of a signaling defe ct may contribute to the ameliorative effect of 7-integrin transgene expression and prevent muscle wasting. One possible mechanism leading to muscle at rophy is through interr uption of the DGCs interaction with the ex tracellular matrix and lo ss of cellular signaling through this complex (142). The DGC comprises of two dystroglycan protein subunits, and -dystroglycan is a transmembrane protein that binds to dystrophin in the cytoplas m and interacts with Grb2, an adapter protein linking signaling molecules containing phosphotyros ine residues and proline-rich domains (205), and FAK, anothe r protein involved in transmembr ane signaling (208). Both Grb2 and FAK function as mediators of survival si gnaling in numerous cell types, often working through PI3K and one of its downstream effectors, a serine/threonine kinase known as Akt (164). Cell adhesion to the extracellula r matrix activates lipid-associated PI3K (26), which in turn induces translocation of Akt from the cytopl asmic pool to the cell membrane, where it is activated by phosphorylation (41). Akt activation ha s a protective effect, blocking induction of apoptosis in differentiated muscle cells in response to disruption of dystroglycan-laminin interactions (107). Downstream elements of th e PI3K/Akt signaling pathway that may mediate cell survival include GSK-3 members of the GLUT family of glucose transporters, transcription factors in the Forkhead family (FOXO), and members of the I B family. Overview of Akt Pathway Contribution to Atrophy Akt affects transcription and translation of through m odulatio n of a number of factors including mTOR, p70s6k, and NFB (Figure 2-3). Inhibition of PI3K reduces Akt and GSK-3

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23 phosphorylation and decreases myotube survival Disruption of the dystroglycan-laminin interaction by a blocking antibody for -dystroglycan significantly reduced Akt and GSK-3 phosphorylation suggesting these signals are modulated by DGC interactions with the extracellular matrix. A retroviral infection with a cDNA c onstruct expressing a constitutively active form of Akt in combina tion with the blocking antibody restores GSK phosphorylation and promotes cell survival (107). Various models of atrophy all lead to upre gulation of the muscle specific E3 ligases, MAFbx and MuRF1 (19, 154) but can be antagon ized by simultaneous treatment with IGF-1 (149) acting through the PI3K/A kt pathway (154, 166). This s uggests a novel role for Akt inhibition of atrophy signaling. The mechanis m by which Akt inhibits MAFbx and MuRF1 upregulation involves the FOXO family of transcription factors (110, 154, 166). FOXO transcription factors are excluded from the nucleus when phosphorylated by Akt and translocate upon dephosphorylation. The translocation of FOXO is required for upregulation of MuRF1 and MAFbx and is sufficient to indu ce atrophy (159). Another important substrate of Akt to note is glycogen synthase ki nase 3 beta (GSK-3 ), which is a negative regul ator of protein synthesis. GSK-3 s activity is inhibited by phosphorylation by Akt. A role for GSK-3 in NFB signaling pathways of disuse atrophy will be discussed in later sections. Disruption of DGC during Unloading Evidence suggests that the DGC wor ks as a re gulator of muscle atrophy and serves as a scaffold for anti-atrophic signal transduction (169). The forced expression of dystrophin in skeletal muscle of cachexic mice is sufficient to attenuate muscle wasting (2). However, in tailsuspension-induced atrophy the e xpression patterns of the com ponents of the DGC, dystrophin, -dystrophin, -sarcoglycan, dystrobrevin, laminin2, 1-syntrophin, and caveolin-3 are

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24 normally expressed at the sarcolemma. Importantl y, nNOS dissociates and translocates to the cytoplasm of the muscle during disuse (2). Norm ally, nNOS is present under the sarcolemma and accumulated at the NMJ (33) and at the myotendinous junction (32) (Figure 24). It is associated with the complex of DAPS and DAGs and with dystrophin through the intermediary of syntrophins, via a PDZ domain ( 194) (Figure 2-2). Neuronal NOS is a peripheral member of the DGC and may play a role in transducing mechan ical signals into chemical ones. Sarcolemmal associated nNOS is reported to be a necessary signaling molecule for satellite cell activation, glucose uptake, muscle contraction, and vasodi lation (7, 52, 68, 97). However, upon dissociation from the DGC, nNOS generates an overabundance of nitric oxide leading to skeletal muscle atrophy (2). Disruption of -sarcoglycan or dystroglycans diss ociates the DGC, including nNOS, and can mimic models of muscular dystrophy. Howe ver, during disuse atrophy both of these molecules remain bound to the sarcolemma wh ile nNOS dissociates from the DGC. This suggests that localizatio n of nNOS may be the key componen t of the DGC that impacts muscle maintenance and atrophy. Nitric Oxide Contribution to Atrophy Nitric oxide (NO) is a gaseous free radical prom oting many biological effects. Due to its high chemical reactivity, NO can be a powerful signaling molecule and an tioxidant or can be harmful through the nitrosylation of many prot eins. NO is generated exclusively by three NO synthase isoforms. Two of them ar e constitutively expressed in cel ls and have been identified as neuronal NOS (nNOS) and endothelial NOS (eNOS). The expression of the third form, inducible NOS (iNOS), is induced by various cytokines. All thr ee isoforms catalyze the formation of NO from arginine, oxygen, and NADPH. Cofactors also required for NOS activity include, tetrahydrobiopterin (BH4), FAD and FMN, in addition to a heme prosthetic group. To acquire the

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25 active state, nNOS and eNOS also require calmodulin (CaM) and Ca2+ ions, indicating that NO synthesis is triggered by an elevation of free [Ca2+]i. Conversely, iNOS is insensitive to Ca2+ ions due to its high affinity for th e CaM binding site at basal Ca2+ levels. A number of structural differe nces characterize the three NOS isoforms. At the N-terminal region, nNOS contains a PDZ domain through which it interacts with 1-syntrophin in the membrane cytoskeleton-dystrophin complex of skel etal muscle or with postsynaptic density proteins in synaptic membranes and neuromuscular junctions. This segment is absent in eNOS and iNOS. The localization of each NOS isoform in skeletal muscle varies as shown in Figure 24. The myotendinous and neuromuscular junctions ( 32), like the costameres are rich in nNOS, especially in type II fibers (69). Endothelial NOS is expressed in the cytoplasm of all fibers, but more abundantly in types I and IIa (76), where it is mainly localized in mitochondria (98). Inducible NOS is present at very low levels in healthy skeletal muscle of rodents (177) but is enhanced in response to endotoxin (177). The presence of iNOS is either cytosolic or concentrated at the neur omuscular junction (206). The two constitutive isoforms, nNOS and e NOS, produce pico-nanomolar amounts of NO; whereas, iNOS tends to release NO at nano-micromolar concentrations (161). The quantity of NO produced in skeletal muscle may be a testamen t to the favorable versus unfavorable effects of NO signaling. The lower concentrations of NO produced may have more beneficial effects including satellite cell activation (7), enhanced myotube fusion (118), and muscle fiber hypertrophy (162, 163); whereas, higher amounts of NO, such as those produced by iNOS, are implicated in pathophysiology of inflammatory conditions and degenerative diseases. As mentioned previously, greater concentrations of endogenous NO can result in the formation of

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26 several reactive nitrogen species causing cellular injury due to increased lipid peroxidation and nitrosylation of proteins. Evidence indicates that NOS activity is incr eased in immobilized muscle, resulting in altered production of NO (100). Suzuki and colleague s demonstrate that nNOS is dislocated from the sarcolemma during hindlimb unloading leading to an increase in th e production of NO (169). Neuronal NOS null mice suffered much milder atrophy, decreased nuclear accumulation of FOXO3a, and prevented upregulation of MAFbx and MuRF1 (169). This data indicates that dislocation of nNOS from the DGC increases nNOS production of higher than healthy muscle and mediates muscle atrophy via regula tion of FOXO transcription factors. Conversely, nNOS expression and activity ha s also been shown to increase with mechanical activity (178), traini ng (10), and electrical stimulation (145). NOS is also necessary for muscle hypertrophy (162). Tidball and colleagues show that after 10 days of unloading nNOS protein and mRNA expression drop only to return to control levels after 7 days of reloading (178). NO release was not measured in the unloaded or reloaded muscles. However, after passive stretch and electrical stimulation of soleus muscles and C2C12s, NO release increases 20% above control levels (178). More recently,Wozniak and cohorts demonstrated that a 10% mechanical stretch applied to primary cultured myotubes transiently increased fluores cence of the NO probe, DAF-2DA; whereas in myotubes from mdx mice, wh ich contain little nNOS at the sarcolemma, exhibited much less NO release afte r stretch (201). These results s uggest that mechanical loading is a positive regulator of NOS expression and activity in myotubes and fully differentiated muscle, and this response in essential for muscle growth. If elevated NOS activity is essential for both skeletal muscle hypertrophy and atrophy, what is the mechanism of its effects? The answer to this question is likely related to intracellular

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27 NOS localization, NO concentration and its effect on redox balance, and NO targets in skeletal muscle. Agarwal and colleagues have demonstrated using cyclic stretch in chondrocytes that moderate amounts of strain increases NO produc tion by about 20%, which is the same induction that Tidball measured in muscle cells with res ponse to loading stimuli. The moderate cyclic stretch (8-10%) di d not induce NFB translocation; however, more intensive strain (15-18%) greatly induced NO production (100% above control levels) and induced NFB nuclear translocation. Interestingly, a cytokine challenge induced translocation of NFB, but after initiating moderate cyclic strain (6%), NFB became progressively more cytosolic. This suggests that low levels of NO produced during activity contri bute to maintenance of redox balance, inhibiting NFB activity, and suppressing atrophy signaling pathways. Whereas, high levels of NO induce oxida tive stress, activate NFB, and contribute to turning on atrophy signals. Secondly, the targets of NO in skeletal muscle vary based on its concentration. The main protein targets in vivo include Ca2+-ATPase, the ryanodine receptor-calcium release channel and guanylate cyclase. The ryanodine receptor and Ca2+-ATPase are susceptible to S-nitrosylation which increases open channel probability ( 203) and decreases calcium uptake (191), respectively, at rela tively high concentrations of NO (167). This may disrupt calcium homeostasis in muscle and activate the calcium-d ependent proteases discussed earlier. On the other hand, at lower concentrations, NO activat es guanylate cyclase and produces cGMP (8), which has proven to be an essential signaling path way in skeletal muscle to increase glucose transport (209) and control myoblast fusion (138). The explanation of nitric oxides effects on cellular signaling may be two-fold. First, we hypothesize that activity-induced ni tric oxide production (moderat e levels) contributes to the

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28 maintenance of redox balance and primes the cell for optimal signaling and potential for hypertrophy and adaptation. Second, reduced activ ity causes nNOS dissociation from the DGC leading to high NO production, a shift in redox toward oxidative stress, and activation of proteolysis and atrophy signaling (Figure 2-5). Nuclear Factor of B Pathw ay of Atrophy NFB is a long known signaling molecule of musc le atrophy, and there are five identified NFB transcription factors (p65, Rel B, c-Rel, p52, and p50) that mediate a variety of processes. All family members are expressed in skeletal muscle. Activation of NFB is achieved by nuclear transport of heterodimers of NFB family members and often occurs by ubiquitination and degradation of the inhibitory protein I B, which otherwise binds NFB heterodimers and retains their cytosolic residence (9). NFB members p50 and p52 can form homodimers (75) and undergo nuclear translocation following partial processing of their cytoplasmic precursor molecules, p105 and p100, respectively. Genera lly, p50 and p52, which lack transactivation domains, function as transcrip tional repressors. However, upon binding with B cell lymphoma 3 (Bcl-3), an I B family member, these complexes can activate transcriptio n through the Bcl-3 transactivation domain (22, 57, 133) when Bcl-3 becomes phosphorylated (25, 129). Thus, Bcl-3 is an unusual member of the I B family because it can function as a transcriptional coactivator. Activation of NFB is required for muscle degradation due to TNFtreatment; however, the classical pathway that is activated due to TNFappears to be different than the NFB pathway that is activated during unloading atroph y (Figure 2-6). The classical pathway involves the nuclear transport of p65/p50 he terodimers by degradation of I Btriggered by its phosphorylation by I B kinase (IKK ). NO production by iNOS has been shown to induce this classic pathway (5, 12). However, with unloading, Hunter colleag ues demonstrated that nuclear

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29 levels of the prototypical NFB family member p65 were not el evated, but p50 and Bcl-3 were markedly increased (84). Although both the classi cal and alternative path ways to muscle loss converge at the ubiquitin-proteasome (109, 170), the NFB pathway activated in inactivity is clearly distinct from that found in cachexia and injury. Hindlimb suspension of NFB1(-/-) (p105/p50) and Bcl-3(-/-) mice further marked the necessity of p50 and Bcl-3 for skeletal musc le disuse atrophy. These genetic knockouts abrogated all NFB transcriptional activity and soleus fiber atrophy following 10 days of unloading (83). Judge and colleagues have al so shown that inco rporation of the I B super repressor into mice skeletal musc le reversed muscle atrophy, NFB activity, and significantly reduced FOXO3a and MAFbx expression after 7 da ys of unloading (91). Further, mice with muscle-specific expression of a constitutively active form of IKK and given the proteasome inhibitor MG-132 blocked the increased proteolysis suggesting that NFB activation alone can activate proteasome-dependent prot eolysis (31). Taken together, these results demonstrate the requirement of NFB signaling, specifically through p50 and Bcl-3, in disuse muscle atrophy. A potential link between NO signaling and NFB lies within the nuclear p50/Bcl-3 complex required for disuse atrophy signaling. Bcl3 is necessary for p50 dimer transcriptional activity since it contains the transactivati on domain. However, phosphorylation of Bcl-3 is required prior to its activation. One likely ki nase implicated in Bcl-3 phosphorylation and activation is GSK-3 Our lab has demonstrated that low levels of NO can inhibit GSK-3 dephosphorylation and translocation to the nucle us via PI3K/Akt signaling (Drenning et al. unpublished). Inhibi tion of GSK-3 translocation would there by prevent activation of the p50/Bcl-3 complex and subsequent tran scription of atrophy genes. The NFB pathway is highly

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30 implicated in the progression of disuse skeletal muscle atrophy, and strate gies to attenuate its activity could provide potential therapeutic benefits. Summary Disuse skeletal m uscle atrophy is mainly due to an increase in proteolysis. Lack of mechanical strain on the sarcolemma with inactivity may cause nNOS dissociation from the DGC and reduce PI3K/Akt activity thereby inducing FOXO3a and GSK-3 nuclear translocation and upregulation of the E3 ligases (MAFbx and MuRF1) and NFB (p50/Bcl-3), respectively. Movement of nNOS from the membrane to the cy tosol initiates altered NOS activity potentially leading to oxidative stress a nd further activation of proteo lytic pathways. Normal nNOS localization and mechanical activ ity can produce beneficial effect s in muscle, attenuate GSK-3 through a cGMP dependent mechanism, and suppress NFB activation. Both NOS and NFB play a role in redox balance, have been deemed necessary for disuse skeletal muscle atrophy and found sufficient to induce FOXO3a and E3 ligase expression. Therefore, determining if nitric oxide can prevent and/or contribute to sk eletal muscle atrophy by regulating NFB signaling is important and may lead to potential therapeutic strategies to alleviate skeletal muscle disuse atrophy.

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31 Figure 2-1. Contributions to skeletal muscle protein loss during unloading.

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32 Figure 2-2. Model of the dystroph in-glycoprotein complex as a transsarcolemmal linker between the subsarcolemmal cytoskeleton and the extracellular matrix.

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33 Figure 2-3. Overview of PI3K/Akt signali ng network. Note the link between membrane receptors and Akt control of atrophic pathways.

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34 Figure 2-4. Subcellular compartm entalisation of NOS isoforms in skeletal muscle. nNOS is present below the plasma membrane and is particularly accumulated at the muscular and myotendinous junctions. eNOS is present in the cytoplasm of muscle, principally in the mitochondria. Lastly, iNOS is present in the muscle cytoplasm.

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35 Figure 2-5. Degree of skeletal muscle atr ophy as a function of NO production during various conditions.

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36 Figure 2-6. Pathways of NFB signaling in cachexia/cytoki ne-induced muscle atrophy (classical pathway) versus disuse muscle atrophy.

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37 CHAPTER 3 MATERIALS AND METHODS Experimental Designs Five experim ents were performed in order to develop an in vitro model of disuse muscle atrophy and to determine the role of nitric oxide in two differe nt types of skeletal muscle atrophy. The first three experiments tested if cessation of repetitive cyclic mechanical strain can be used to mimic disuse muscle atrophy as seen in vivo The final two expe riments assessed if high mechanical strain is sufficient to induce the classical pathway of atrophy as seen in cachexia and inflammatory models. Because many complex pathways have been im plicated in skeletal muscle atrophy (e.g., Akt, NFB, calpain, caspase), we chose to study some of the main players implicated in several in vivo studies to determine th eir importance during our in vitro model of myotube atrophy. Our study incorporated two separate methods of in vitro culture. The first is an immortal mouse cell line of skeletal muscle cells called C2C12s. These cells were originally obtained by Yaffe and Saxel through selective serial pa ssage of myoblasts cult ured from the thigh muscle of C3H mice 70 h after a crush injury (204). Th ese cells are capable of differe ntiation and are a widely used model to study differentiated skeletal muscle ce lls. Our second model, termed primary culture, involves isolating satellite cells from mouse sk eletal muscle and inducing myoblast lineage under appropriate medium and growth factor conditions. Primary cultured cells play a role as a bridge between cell lines and cells in vivo and this technique allowed us to utilize transgenic mouse strains to elucidate the signals that are dependent upon NOS gene expression during atrophy of myotubes. Experimental 1 Design Ai ms 1 and 2 (Figure 3-1). Experiment one was used to test Specific aims 1 and 2. C2C12 myotubes were subjected to a 12% cyclic stretch (1h/d; 1 Hz) for

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38 two days (2dSTR) or five days (5dSTR). One group underwent two days of activity (stretch) followed by three days of inactivity ( 2dSTR3dCES) to induce myotube atrophy. A nonstimulated control group was used at each time point to control for myotube maturation (2dCON and 5dCON). To test hypothesis one, images were taken of each group at the end of the experimental period and analyzed for myotube length, diameter, a nd area. Whole cell lysate was collected and used to assess total protein content and protein degradation. To examine hypotheses 2A and 2B, culture medium was collect ed at 48 h intervals to test for NO production by means of the nitrate/nitrite fluorometric k it (Cayman Chemical). Three samples from each group were fixed and immunostained against nNOS to confirm its localizati on within the cell. To test hypothesis 2C, L-NAME (5 mM) was administ ered in parallel to all groups to assess NO regulation of myotube atrophy. Experimental 2 Design Aim 2 (Figure 3-2). C2C12 myotubes were subjected to a 12% cyclic stretch (1 h/d; 1 Hz) for two days and then remained inactive for 1, 12, 24, or 48 h before harvesting for protein or luciferase activity. We chose to use a 12% magnitude stretch based on results that that a moderate degree of cyclic st retch stimulates an approximately 10-20% increase in NO production (5, 178). We confirmed these NO levels by measurement of the NO probe, 4amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate, at 6, 12, 18% stretch (Figure 3-3). The aforementione d time points were chosen to test for a time course of upregulation of atrophy signaling pathways. According to our preliminary data, one hour of NO treatment was effective in altering th e phosphorylation states of Akt, GSK-3 FOXO3a, and nuclear translocation of Bcl-3; thus two bouts of a one hour stre tch were chosen to test for changes in these proteins. L-NAME (5 mM) was ad ministered 30 m prior to the first bout of

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39 stretch and maintained in the medium throughout the experimental period to test for NOdependent activation of all measures. In experiment 2a, we examined the upre gulation of atrophy signaling molecules during the cessation of stretch by assessing whole cell lysa te as well as nuclear and cytosolic fractions by western blot to probe for the phos phorylation status of Akt, GSK-3 FOXO3a and Bcl-3 and nuclear and/or cytosolic localization of the NFB members, p50, p65, Bcl-3, and I B. In experiment 2b, we monitored NFB-dependent transcriptiona l activity in myotubes by transfection with a NFB-luciferase reporter plasmid. Experimental 3 Design Aim 2 Hypothesis 2C (Figure 3-4). Primary cultured satellite cells from wild-type (WT) or nNOS(-/-) mice were harvested and plated to 100 mm culture dishes. However, our technique was unsuccessful in ma intaining these satellite cells in culture. Therefore, we were unable to collect data for hypothesis 2C in Specifc aim two which was to subject primary cultured myotubes to 12% cyclic stretch (1 h/d; 1 Hz) for 2 d with cessation of stretch for 1, 12, 24, or 48 h before harvesting for protein as described in experiment two. Experiment 4 Aim 3 (Figure 3-5). C2C12 myotubes were subjected to a high magnitude stretch (18%; 0.1 Hz) for 1, 2, 3 h. One group then remained inactive for 2 h following the stretch protocol (2hCES). High magnitude stretch (18-26%) has b een shown to upregulate iNOS mRNA and induce NFB translocation in other cell types (5). According to our preliminary data, 18% stretch is sufficient to induce a 30% increase in NO production (Figure 3-3). Although 30% increase in NO is sufficient to induce iNOS mRNA and NFB translocation, higher amounts of NO have shown to produce the greatest e ffect (5) but also may cause detrimental and undesirable effects to the cell. T hus we chose to implement an 18% cyclic strain to skeletal muscle myotubes as a model for injury a nd to induce the classical pathway of NFB signaling.

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40 The aforementioned time points were chosen to test for a time c ourse of upregulation of iNOS mRNA and NFB translocation previously shown in ot her cell types by Ag arwal and colleagues (5). In experiment 4a, we examined the translocation or degradation of NFB family members associated inflammation and injury in the cla ssical pathway of atrophy by isolating nuclear and cytosolic fractions and analyzing with western blots to probe for the nuclear localization of the NFB members, p50, p65, Bcl-3, and cy tosolic content of p65 and I B. MAFbx was used as an indirect measure ubiquitin-proteasome activity and protein degradation. In experiment 4b, we monitored NFB-dependent transcriptional activity in myotubes by transfection with a NFBluciferase reporter plasmid. In experiment 4c, we isolated mRNA and performed real-time RTPCR for iNOS expression. Experimental 5 Design Aim 3 Hypothesis 3B (Figure 3-6). Primary cultured myotubes from wild-type (WT) or iNOS(-/-) mice were subjected to a high magnitude stretch (as determined from experiment 4) for 1, 2, and 3 h. One group then remained inactive for 2 h following the stretch protocol (2hCES). Measures for experiments 5a were identical to those in 4a. General Methods Myogenic Culture Myoblasts derived from C2C12 cells (ATCC, Manassas, VA) were cultured on 100 mm dishes in Dulbecoos Modified Eagles Medium (DMEM) (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (F BS), 1% penicillin/streptomycin, and 0.1% funzigone at 37 C in the presence of 5% CO2 until 50-60% confluence was reached as visualized by light microscopy. The cultures were then be tr ypsinized and replated at equal density to 6-

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41 well Flexcell plates for mechanical stimulati on as described below. Once the Flexcell plate cultures reach 60-80% conf luency, myotube differentiation was initiated by switching to DMEM supplemented with 2% horse serum, 1% pe nicillin/streptomycin, and 0.1% fungizone. Primary Culture Muscles from both hindlim bs from 3 mice (nNOS(-/-), iNOS(-/-) or their respective wildtypes) were pooled and satellite cells were isolated according to Pavlath et al (136). Myogenic cells were purified by preplating and seeded in 100 mm dishes cultured in Hams F-10 with 20% FBS, 5 ng/mL basic FGF, 1% penicillin/strept omycin, and 0.1% fungizone Once cells reached 80% confluence, they were trypsinized and replat ed to Flexcell plates under the same conditions as described above. Mechanical Stimulation Using Cyclic Strain Myoblasts were plated on type I collagen-coated flexible-bottom plates (Bioflex plates, Flexcell International, McKees port, PA) and incubated at 37 C in an incubator maintained at 5% CO2 until 90% myotube population was visualized. Imme diately prior to the initiation of stretch, the medium was aspirated and changed to differe ntiation medium supplemented with or without 5 mM L-NAME. The cells were subjected to cycl ic strain at 1-0.1 Hz (1 s of 4-18% stretch alternating with 1-10 s of relaxation) for 1-4 h using a computer-controlled vacuum stretch apparatus (FX-4000T Tension Plus System, FlexCell International) with a vacuum pressure that is sufficient to generate to predetermined percentage of mechanical strain. Replicate control samples and cells undergoing cessation of stretch we re maintained under stat ic conditions with no applied cyclic strain. The Flexcell system used for our stretch appa ratus employed equibiaxial deformations to the membranes on the bottom of each culture plate. Equibiaxial stretch is the preferred model of in vitro deformation because a myotube adhered in any direction is subject the same shape

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42 change. Although the Flexcell Bioflex software uses percentile stretch to calibrate the vacuum necessary to deform the elastic membrane, each cell is not exposed to the same percent of stretch. The percent stretch woul d depend on the orientation and length of the myotube. Larger myotubes would be stretched a smaller percent th an those of a lesser size. Specifically, the 12% stretch used in aims one and two corresponded to a 4.2 mm stretch equal in all directions, and the 18% stretch used in aim three referred to 6.3 mm deformation. While the orientation of the myotube on the membrane would still affect the direction of deformation, the myotubes had a tendency to align in the same plane with ma turation and exposure to stretch as observed previously (39). In addition, the myotubes adhere d to the section of the membrane that was forced over the loading post during stretch expe rienced greater stress a nd often detached from the plate in comparison to myotubes at the cente r of the well. Thus, these peripheral myotubes were excluded from all images and immunostaining. Immunohistochemistry Myotubes w ere fixed in 2% paraformaldehyde for 10 min, washed twice with PBS, and membranes were permeabilized for 15 min with 0.2% Triton-X. Myotubes were blocked in 5% goat serum for 30 min, followed by incubation in primary antibody diluted in 0.5% BSA for dystrophin (Lab Vision Corporat ion), MHC type IIa (1:50; N2.261 Developmental Studies Hybridoma Bank) nNOS (1:133; BD Transduc tion Labs), eNOS (1:133; BD Transduction Labs), or iNOS (1:133; BD Transduction Labs ) for 1 h. Myotubes were then incubated in secondary antibody diluted in 5% Pierce Super Blocker (Pierce Biotechnology) for Rhodamine (1:40, Invitrogen) or Alexa 488 IgG (1:133, Invi trogen) for 1 hour and stained with DAPIcontaining mounting media (Vec tor Laboratories). All culture s were viewed on a Zeiss microscope with rhodamine, FITC, and DAPI filters.

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43 Image Analysis Three dig ital images per culture were captured using a Zeiss microscope. The images were analyzed for myotube length, diameter and ar ea using ImageJ imaging software (NIH). Nuclear and Cytosolic Fractionation Cells were harvested in cytosolic extr action reagent (CERI; Pierce Biotechnology) containing 0.05% vol/vol proteas e inhibitors and 0.5% vol/vol phosphatase inhibitors from Sigma and centrifuged, and the resulting pellets was treated with NE-PER nuclear and cytosolic extraction reagents according to the manufacturers procedures (Pierce Biotechnology, Rockford, IL). The nuclear fraction was confirmed by wester n blot for histone H2B (Upstate; Lake Placid, NY) and cytosolic fraction for -actin (Abcam; Cambridge, MA). Whole Cell Lysate Cells were harvested in ice-cold non-denatu ring lysis (NDL) buffer containing 30m M TrisHCL (pH 7.5), 0.7% Triton-X, 150 mM NaCl, 3.5 mM EDTA, 10 mg/ml NaN3, 1 M Na3VO4, 0.05% vol/vol protease inhibitors and 0.5% vol/vol phosphatase inhibitors (Sigma, St. Louis). Lysates were then centrifuged at 4C for 10 minutes at 1000 x g. Western Blot Analysis Protein concentrations w ere measured using the DC Protein Assay Kit (Bio-Rad Laboratories, Richmond, CA). Aliquots of whole cell lysate or nuclear and cytosolic samples were ran on SDS-PAGE gels and proteins were transferred to nitrocellulose membranes and blocked with Odyssey blocking buffer for 1 h. The membranes were incubated at 4C overnight in primary antibody diluted with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE), TBS, and 0.01% Tween-20 for phospho-GSK-3 (Santa Cruz; Santa Cruz, CA), total GSK (Santa Cruz; Santa Cruz, CA), phospho-AKT (Santa Cruz; Santa Cruz, CA), total AKT (Santa Cruz; Santa Cruz, CA), p65 (Abcam; Cambri dge, MA), p50 (Abcam; Cambridge, MA), I B-

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44 (Santa Cruz; Santa Cruz, CA), Bcl3 (Santa Cruz; Santa Cruz, CA), -actin (Abcam; Cambridge, MA), and histone H2B (Upstate; Lake Placid, NY). Membranes were washed with TBS-T three times and incubated for 35 min in secondary antibody, Odyssey blocking buffer, and TBS-T. The secondary antibodies were IR Dye conjugated secondaries obtained from LICOR detectable at wavelengths of 680 or 800 nm. Membranes were washed three times with TBS-T and once with TBS before being scanne d and detected using the Odyssey infrared imaging system (LI-COR). Isolation of RNA and Real-Time RT PCR Myotubes w ere washed in ice-cold PBS and harvested in TRizol (Invitrogen; Carlsbad, CA) for RNA isolation. Quantifi cation of mRNAs were performed with specific primer and probes using TaqMan technology designed by Applied Biosystems for quantitative real-time PCR. Briefly, concentration and purity of the extracted RNA were measured spectrophotometrically at 260 and 280 nm absorbance in 1X Tris-EDTA buffer (Promega, Madison, WI). RT was performed using the Supe rScript III First-Strand Synthesis System for RT-PCR according to the manufacturers instructions (Life Technologies, Carlsbad, CA). Reactions were carried out using 1 g of total RNA and 2.5 M o ligo(dT)20 primers. Firststrand cDNA was treated with two units of RNase H and stored at 80C. Primers and probes were obtained from Applied Bios ystems: iNOS (GenBank NM_010927.3, assay Mm0040485_m1); GAPDH (GenBank NM _008084.2, assay Mm99999915_g1). Primer and probe sequences from this service are proprieta ry and therefore are not reported. Quantitative real-time PCR for the target genes were perf ormed in the ABI Prism 7700 Sequence Detection System (ABI, Foster City, CA), using the 2CT method, where CT is threshold cycle, and glyceraldehyde-3-phosphate dehydrogena se (GAPDH) as the control gene.

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45 Nuclear Factor of B-Dependent Transcriptional Activity NFB activ ity was assessed using a transient transfection of a reporter plasmid. Myoblasts were transfected with either a reporter plasmid c ontaining the firefly lucife rase gene driven by a promoter sequence containing five repeats of a consensus NFB binding site or a negative control plasmid (pNFB-luc or pCIS-CK, Stratagene; 2 g/w ell). Cells were cotransfected with a second plasmid (pRL-CMV, 0.04 g/well; Promega, Madison, WI) to control for transfection efficiency. Plasmids were complexed with Lipofectamine reagent (Invitrogen) and exposed to myoblasts in Opti-MEM for 6 h. After transfectio n, cells were placed in 6% HoS in Opti-MEM for 18 h before being switched to differentiati on media (DMEM supplemented with 2% HoS and 1% penicillin-streptomycin). Differentiation me dium was refreshed every 48 h until confluent myotubes were formed (5 days). Dual Luciferase Assay Imm ediately following treatment, myotube cultur es were washed with ice-cold PBS and lysed by addition of 500 l passive lysis buffer (Promega). Plates were rocked at room temperature for 15 min. The lysate was then tran sferred to microcentrif uge tubes and centrifuged for 30 s at 400 x g to sediment cellular debris. Fi refly luciferase (origina ting from transcriptional activity of the pNFB-luc or pCIS-CK vectors) and renilla luciferase activities were measured sequentially in the same 10 l volume of cell lysate using the dual luciferase assay kit (Promega) according to the manufacturers instructions and a luminomete r (model FB12, Berthold) set to measure average light intensity in relative lig ht units (RLU) over a 10-s measurement period. NFB-dependent transcriptional activity for each samp le was taken as the raw firefly luciferase activity (RLU) divided by the renilla luciferase activity (RLU). For each experiment, all values are expressed relative to the average of the control group.

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46 Nitric Oxide Production Intracellular NO was m onitored with DAF-FM diacetate (Invitrogen, Carlsbad,CA), a pHinsensitive fluorescent dye that emits increa sed fluorescence after reac tion with an active intermediate of NO formed during th e spontaneous oxidation of NO to NO2. Myotubes were incubated at 37C for 30 min in phenol red-free, serum-free DMEM containing 10 M of DAFFM diacetate. After loading is completed, cells were rinsed three time s with phenol red-free, serum-free DMEM and subjected to 1 h of mechanic al strain. Cells were harvested in deionized water, centrifuged at 12,000 x g and aliquots of 100 l were quantified on a microplate fluorometer (Molecular Devices) using excitation and emission wavelengths of 488 and 520 nm, respectively. NO production in the medium was a ssessed with the Nitrate/ Nitrite Fluorometric Assay Kit from Cayman Chemical (Ann Arbor, MI). This assay measures the sum of nitrate and nitrite, the final products of nitric oxide metabolism, in a tw o-step process. Fi rst, nitrate is converted to nitrite using nitr ate reductase. This is followed by reaction of nitrite with 2,3diaminoaphthalene to produce the fluorescen t compound, 1(H)-naphthotriazole, which is quantified on a microplate fluorometer (Molecu lar Devices) using excitation and emission wavelengths of 360 and 430 nm, respectively. Statistical Analysis Group sam ple size was determined with power analysis of our preliminary data. Comparisons between groups were made by a 3-way full factorial ANOVA, and when appropriate, Tukeys HSD test wa s performed post-hoc. Signifi cance was established at p<0.05.

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47 Figure 3-1. Experiment 1 design for Aims 1 and 2. Purpose was to develop an in vitro model of disuse skeletal muscle atrophy with cyclic stretch and test wh ether NOS inhibition prevented the progression of atrophy.

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48 Figure 3-2. Experiment 2a and 2b designs for A im 2. Purpose was to monitor the activity of members of the Akt and NFB pathways during cessation of stretch and test whether NOS inhibition prevented their activity.

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49 Figure 3-3. Average 4-amino-5methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate fluorescence in C2C12 myotubes after 1 h of cyclic stretch at various magnitudes.

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50 Figure 3-4. Experiment 3 design for Aim 2. Purpose was to monito r the activity of members of the Akt and NFB pathways during inactivity us ing genetic and pharmacological inhibition of nNOS in primary cultured myotubes.

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51 Figure 3-5. Experiment 4 design fo r Aim 3. Purpose was to monitor activity of the classical NFB pathway during high magnitudes of stre tch and test whether NOS inhibition altered its activity.

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52 Figure 3-6. Experiment 5 design fo r Aim 3. Purpose was to monitor activity of the classical NFB pathway during high magnitudes of stre tch using genetic and pharmacological inhibition of iNOS in primary cultured myotubes.

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53 CHAPTER 4 RESULTS Skeletal Muscle M yotube Atrophy Model The purpose of Specifc aim 1 is to develop models of atrophy in vitro using skeletal muscle myotubes. The hypothesis that cessation of a daily cyclic stretch will cause myotube atrophy is based on the concept that daily repetitive stretching of the muscle membrane induces protein synthesis (81), muscle metabolism (86, 155, 178), NO production (178, 212), and muscle integrity (213) via mechanotransduction pathways Withdrawal of the st retching stimulus is sufficient to cause significant remodeling, pr otein degradation, and myotube atrophy. Our protocol used the Flexcell TensionPlus system by which skeletal muscle myotubes are plated on flexible bottom plates and submitted to a vacuum generated stretch for 1 h/d for 2 d at a 12% strain (0.7 Hz). The 2 d of a cyclic stretch is a mimic of skeletal muscle activity. Following the 2 d of repetitive stretch, the myotubes were re moved from the Flexcell baseplate and remain inactive (i.e. no stretch) for three subsequent days Three days of inactivity is sufficient to cause visual myotube atrophy (Figure 41 and Figure 4-2) and cytoskelet al protein degradation (Figure 4-3 and Figure 4-4). In order to compare treatm ents, cells were divide d into five groups. Two groups were non-stretched controls at the 2 d and 5 d timepoints to control for myotube maturation (2dCON and 5dCON, respectively). One group was stretched for 2 days only with no inactivity period (2dSTR). One group was stretched for the entire 5 day period (5dSTR). The final group encountered 2 d of cyclic stre tch and 3 d of inactivity (2dSTR3dCES). An elevation in NO production due to di ssociation of nNOS from the dystrophinglycoprotein complex has been implicated as a cause for skeletal muscle atrophy during disuse (169). To study the effect of NOS activity in our in vitro model, L-NAME was administered in the culture medium to subgroups of myotubes at each timepoint.

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54 Myotube Atrophy Two days of 12% cyclic stretch for one hour per day did not alter av erage length, diam eter, and area in C2C12 myotubes (Figure 4-2). Importantly, cessation of the stretch for three sequential days thereafter cause d a significant reduction in my otube diameter and area in comparison to all control and stretched groups, representing skeletal muscle myotube atrophy. Cells undergoing five days of consecutive stretch experienced a significant increase in diameter and area. However, treatment with the NOS inhibitor L-NAME in all groups led to diameter and area measurements similar to those of atrophied myotubes, which were significantly lower than non-treated cells. Protein Degradation Skeletal m uscle atrophy is predominately due to increased proteolysis leading to a loss of mass. With our in vitro model of atrophy, muscle protein degradation increased in cells undergoing a three-day cessation of activity (Figure 4-3 and Figur e 4-4). AlphaII-spectrin is a cytoskeletal protein that is susceptible to proteolysis by calpain (generating a 150/145 kDa product) and caspase-3 (generating a 120 kDa product) (171). Western blotting for these cleavage products demonstrated th at following three days of wit hdrawal of mechanical activity in vitro there was significant activation of calpain (Figure 4-3A-B). Caspase-3 may also be active and participate in II-spectrin degradati on during the atrophy protocol employed in our study although the elevation of the 120 kDa cleavage product did not reach statistical significance (Figure 4-3C). Another cytoskeletal protein, talin, interacts with the DGC and focal adhesion proteins, such as integrin and vinculin (53, 193), to ge nerate a transmembrane connection involved in mechanotransduction of skeletal muscle. Calpain is known to cleave the in tact 235-kDa talin into 190and 47-kDa fragments (13), th us calpain-induced protein degradation was also measured as

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55 the ratio of concentrations of the 190and the 235-kDa bands by western blot (Figure 4-4). Myotubes withdrawn from stretch for three days showed significantly grea ter talin proteolytic ratio than all untreated control and stretched cells. Inhibition of NOS also increased talin cleavage in 2 d cultures which is in agreement with other studies that NO can prevent calpainmediated talin proteolysis a nd cytoskeletal breakdown in C2C12 muscle cells (99). The integrins, specifically the 1-integrin subunit found in skeletal muscle, provide strength to the linkage between the cytoskeleton and extracellular matrix and may enable muscle to sense changes in the mechanical envi ronment (14). Further, a decrement in 1-integrin expression is linked to reduced NO production (213). Thus we measured 1-integrin in stretched and atrophied myotubes in our model. Five days of 12% cyclic stretch induced a dramatic and significant increase in 1-integrin protein, whereas two days of stretch was not sufficient to cause any change (Figure 4-5). Beta1-integrin le vels in myotubes atrophied for 3 d did not differ from 5dCON but were significantly lower than 5dSTR cells. Treatment of myotubes with the NOS inhibitor L-NAME showed a trend toward reduced 1-integrin expression in stretched myotubes, but this effect did not reach statistical significance. Although our data provides evidence for an in crease in proteolysis with cessation of stretch, there was no decrement in protein conten t in myotubes after withdrawal from activity (Figure 4-6) and no change in pr otein concentrations with stre tch. Treatment of myotubes with L-NAME only significantly reduced protein c ontent in the 2dSTR3dC ES+LN group. This result is surprising considering that, co llectively, our data indicates that two days of stretch followed by three days of inactivity is sufficient to induce cy toskeletal protein degradation and visual atrophy of C2C12 myotubes.

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56 Cellular Signaling during Disuse The purpose of Specifc aim 2 was to test if cessation of repetitive daily activity of skeletal muscle myotubes causes elevation of NO producti on (>20%) and activation of skeletal muscle disuse atrophy signaling pathways. Specifc aim 2 had three hypotheses. First, we hypothesized that 12% cyclic stretch in C2C12 myotubes would maintain nNOS localization, low levels of NO, and opposes skeletal muscle atrophy. Secondly, ce ssation of stretch would cause dissociation of nNOS from the sarcolemma, elevated NO producti on and initiation of skeletal muscle disuse atrophy signaling. Lastly, our final hypothesis was that NOS inhibition with L-NAME in C2C12 or genetic knockout of nNOS in primary myot ubes would prevent the upregulation of atrophy signaling pathways following ce ssation of cyclic stretch. To test Specifc aim 2, myotubes were differen tiated for 4-5 d before enduring 2 d of 12% cyclic strain for 1 h/d at 0.7 Hz. To determin e the time course for upregulation of atrophying signaling pathways, samples were taken immediatel y after the second bout of stretch (Stretched) and at timed intervals of 12, 24, and 48 h dur ing cessation of stretc h. The non-specific NOS inhibitor L-NAME was administered to subcultures of each group throughout the entire protocol. Measurements in C2C12 Cell Culture during Disuse Myosin heavy chain and dystrophin The resem blance of C2C12 myotubes to adult skeletal muscle is unclear. Thus we sought to determine if C2C12 myotubes contain the cytoskeletal a nd contractile proteins, dystrophin and myosin heavy chain as well as all isoforms of the NOS enzyme. After 6 d of differentiation, immunohistochemistry revealed that C2C12 myotubes do express type IIa myosin heavy chain (Figure 4-7A). This is in agreement with other studies in C2C12s demonstrating that myotubes begin expressing all isoforms of MHC after 3 d of differentiation (40). We did not perform immunostaining on primary cultured myotubes, but they are also capable of expressing all MHC

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57 isoforms. Primary myotubes from rat immunostain for embryonic, neonatal, and type II MHC after 7 d of culture whereas slow isoforms were detected at 12-13 d after plating (182). Dystrophin staining is also evident throughout the C2C12 myotubes (Figure 4-7A). Dystrophin and the glycoprot ein complex with which it is associ ated play a fundamental role in mechanotransduction during stretch and provide st ructural integrity for the cell. Thus the presence of dystropin in normally differentiated C2C12 myotubes was critical for testing our hypotheses of myotube adaptation to cyclic strain and inactivity. Negative controls for each secondary antibody (with no primary antibody) reveal no visible ba ckground fluorescence (Figure 4-7B). Nitric oxide synthase isoforms The presence and localization of the nitric oxide synthases in C2C12s were also unknown. Immunostaining for nNOS, iNOS, and eNOS after 6 d of differentiation re vealed that all three isoforms exist in vitro (Figure 4-8). Both nNOS and iNOS appear to be localized in clusters near the cell membrane whereas eNOS is expresse d more diffusely thr oughout the cytosplasm. Localization of nNOS In norm al adult skeletal muscle, nNOS is local ized at the sarcolemma associated with the dystrophin-glycoprotein complex. Evidence indicates that nNOS is dislocated from the sarcolemma during hindlimb unloading and cont ributes to skeletal muscle atrophy (169). Immunostaining for nNOS in C2C12 myotubes shows nNOS localized in clusters at the cell membrane in all control and stre tched groups (Figure 4-9). However, cessation of activity for 3 d causes nNOS localization to become more diffu se throughout the myotube potentially indicating its dissociation from the sarcol emma and migration to the cyto plasm. Treatment of myotubes with L-NAME does not appear to alter nNOS lo calization during the first 2 d of treatment;

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58 however, after 5 d, nNOS appears to lose its clustering effect and becomes more diffuse throughout the cell (5dLN, 5dSTR+LN, 2dSTR3dCES+LN). Nitric oxide production The final products of nitric oxide in vivo are nitrate (NO3 -) and nitrite (NO2 -) (125). The relative proportion of these products is variable and cannot be predicted wi th certainty. Thus, the best index of total NO production is the sum of both nitrate and nitrite. To test for changes in nitric oxide concentrations in stretched and atrophied myotubes, we measured total nitrate plus nitrate content in the culture medium. Although ni trate concentrations di d not reach statistical significance, there was a trend for an increase in NO levels with stre tch (Figure 4-10). NOS inhibition by addition of L-NAME to the cultur e medium was successful in attenuating the stretch-induced NO production, but did not alter basal levels. NO release in the 2dSTR3dCES group was not significantly different than that of the 5dSTR cells but showed trends to be higher than 5dCON cells and comparable to cells stre tched for 2 d. This suggests that NO production remains elevated during cessation of activity for up to 72 h and may contribute to the signaling events occurring during inactivit y. Myotubes stretched for 5 d ha d higher NOS activity than the remaining groups demonstrating that an increas e in NO production with stretch is maintained with continued activity but can be blunted with L-NAME administration. Components of the Akt pathway The Akt (or protein kinase B) pathw ay is know n to regulate muscle protein synthesis and has recently been implicated as a master controller of skeletal muscle size by also playing a role in skeletal muscle atrophy (111, 154). We sought to determine if the activity of Akt and its downstream targets were altered with 2 d of cyclic stretch followed by 3 d of inactivity. Akt protein expression. Akt activity, as indicated by its phosphorylation status, was increased 5-fold following the second bout of cyc lic stretch (Figure 4-11 ). This elevation was

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59 transient as it returned to baseline after 48 h. Notably, NOS inhibiti on significantly blunted activation of Akt at all time points indicating that phosphorylation of Akt in C2C12 myotubes is NOS-dependent. GSK-3 protein expression. GSK-3 is activated by its dephosphorylation and translocation to the nucleus where is known to trigger numerous transcription factors. Many of its targets are thought to promote transcription of atrophy-related genes (214); thus, we sought to determine if GSK-3 activity was altered in our stretch m odel. Two days of 12% cyclic stretch was successful in inhibiting GSK-3 activity Figure 4-12). However, the ratio of phosphorylated to total GSK-3 decreased over time with cessa tion of stretch. Interestingly, LNAME administration to the media complete ly prevented phosphorylation of GSK-3 until 48 h post-stretch, suggesting that NO is an impor tant signaling molecule for GSK inhibition in vitro Phosphorylated FOXO3a protein expression The FOXO family of transcription factors is involved in the transcription of many genes regulating ubiquitin-proteasome pathway. FOXO3a is thought to specifically induce gene expression of the muscle specific E3 ligases, MAFbx and MuRF1 which are essential for mu scle protein degradation (154). FOXO3a is phosphorylated and inactivated by Akt, and upon dephosphorylation can translocate to the nucleus to bind to DNA. Next, we sought to dete rmine if FOXO3a was activated in our atrophy protocol. Immediately after stre tch, phosphorylated FOXO3a was significantly elevated (Figure 4-13) and tapered off as time elapsed. Nitric oxide synthase inhibition co mpletely abrogated the increase in FOXO3a phosphorylation, again de monstrating the importance of NO in this signaling pathway. MAFbx protein expression. Protein degradation by the 26S proteasome is dependent on ubiquitination by E3 ligases. MAFbx is a muscle -specific E3 ligase that has been deemed

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60 essential for skeletal muscle atrophy through activ ation of the FOXO tran scription factors (154). In an attempt to correlate Akt and FOXO3a activity with MAFbx expression, we measured MAFbx protein levels usi ng our atrophy model in C2C12s. Immediately after stretch, levels of MAFbx protein were not altered but tended to continually incr ease through 48 h of inactivity, which does mimic the decrease in Akt activity and FOXO3a phosphorylation although this trend was not statistically significant (Figure 4-14). Treatment of the myotubes with L-NAME did not affect MAFbx upregulation in co mparison to untreated controls. Altogether, the anabolic effects of the Akt pathway are upregulated af ter 2 d of moderate magnitude cyclic stretch. Cessati on of activity for up to 48 h is su fficient to upregulate atrophic signals through activ ation of GSK-3 FOXO3a, and MAFbx. Alternative NFB pathw ay Various subunits of NFB have been implicated in atr ophy models to be essential for skeletal muscle loss. The primary NFB proteins operating during inflammation and oxidative stress models are p65 and p50 (59, 202); however, with disuse, genetic knockout of p65 was not effective in attenuating atrophy, whereas mice null for the p50 or Bcl-3 gene suffered minimal muscle loss during hindlimb unl oading (83). Thus we measured all of these subunits to distinguish those involved in our in vitro model of disuse atrophy. Nuclear p50 The NFB member p50 undergoes nuclear translocation following partial processing of its cytoplasmic precursor mol ecule, p105, and after forming a homodimer, can bind to DNA. We found that following two bouts of 12% cyclic stretc h, nuclear p50 protein levels were significantly and transiently elevated but returned to baseline after 12 h cessation of stretch (Figure 4-15). NOS inhibition did not affect p50 translocation until after 48 h where it caused a significant augmentation of nuclear p50.

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61 Bcl-3 Generally, homodimers of NFB p50, which lack transactiv ation domains, function as transcriptional repressors. However, upon binding with B cell lympho ma 3 (Bcl-3), these complexes can activate transcription through the Bcl-3 transac tivation domain (22, 57, 133). Bcl3 activity largely depends on is phosphorylation status (25, 129) and phosphorylation of, the p502/Bcl-3 complex can occur via GSK-3 (190). Thus we tested how Bcl-3 phosphorylation and protein expression were a ffected by cyclic stretch and NOS inhibition. The ratio of phosphorylated to total Bcl-3 was not affected with mechanical stimulation or cessation of stretch (Figure 4-16A). However, L-NAME treatment significantly increased Bcl-3 phosporylation. This corr elates with GSK-3 activity data in L-NAME-treated myotubes (Figure 4-12), suggesting that NO may inhibit GSK-3 and thereby attenuate phosphorylation of Bcl-3. Levels of total Bcl-3 protein were significan tly greater after stretch and throughout 24 h of inactivity but returned to baseli ne after 48 h (Figure 4-16B). I nhibition of NOS increased Bcl-3 expression following 24 h cessation of stretch but did not affect protei n levels at any of the other time points measured. Nuclear and cytosolic p65 In agreement with the litera ture (84), p65 levels were not altered with moderate stretch or disuse atr ophy as indicated by nucle ar and cytosolic p65 measurements (Figure 4-17A-C). Interestingl y, translocation of p65 to the nucleus was significantly elevated by NOS blockade im mediately and up to 24 h after stretch. Cytosolic IkB. The NF-k subunit p65 maintains its cyto solic residence through its binding to I B. Upon phosphorylation by I B kinase (IKK), I B is ubiquitinated and degraded by the proteasome which frees p65 to transloc ate to the nucleus. Western blotting for I Bdemonstrates that NOS inhibition ca used a reduction in cytosolic I Bimmediately following and 12 h post-stretch (Figure 4-18). I Blevels returned to control levels after 24 h. This trend

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62 correlates with the appearance of p65 in the nuclear fraction of C2C12 myotubes after stretch (Figure 4-17A). Further, stre tched but untreated cells expe rienced an increase in I Bat all time points. Our data suggests that nitric oxide may be protective against I Bphosphorylation/degradation and p65 release after moderate magn itudes of stretch, which may occur through regulation of an upstream kinase. Nuclear factor of -B transcriptional activity during disuse Nuclear tran slocati on and binding of NFB subunits to B sites on DNA does not always correspond to increased gene transcription since both p50 and p52 homodimers can act as transcriptional repressors due to lack of a tr ansactivation domain. In order to determine if moderate cyclical strain and wit hdrawal of stretch results in NFB-dependent transcriptional activity, C2C12 myotubes were transfected with either a reporter plasmid containing the firefly luciferase gene driven by a promot er sequence containing consensus NFB binding sites (pNFB-luc) or a negative control plasmid (pCIS-CK). No significant differences were detected with any treatment most likely due to our small sample size (Figure 4-19). However, there was a trend for an increase in NFB activity with stretch that decl ined back to baseline over time. Conversely, L-NAME treatment attenuated this rise immediately after 12% stretch, but induced an upward tendency in NFB-dependent transcripti on as time progressed. The trends demonstrated by NFB transcriptional activity follow the pattern of p50 translocation to the nucleus (F igure 4-15), which suggests that p50 is most likely involved in gene regulation using our model of atrophy. Ho wever, whether or not p65 and/or Bcl-3 are involved in this model remains to be determined.

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63 Myotubes transfected with the negative cont rol vector, pCIS-CK, which contains the luciferase gene but lacks the NF-kB-responsive pr omoter sequence, did no t respond to any of the treatments in any of our transfecti on experiments (Figures 4-19 and 4-25). Neuronal NOS Knockout Primary Satellite Cell Culture To elucidate which NOS isoform is involved in the stretch-induced changes of Akt and NFB signaling, we proposed to isolate satellit e cells from mice null for the nNOS gene. However, our technique proved unsuccessful in main taining these satellite cells in culture. From observation only, fewer myogenic cells were pres ent after isolation, a nd these cells did not demonstrate the proliferative qualities seen in other primary cultured my oblasts. Therefore, we were unable to collect data for the final hypothesi s in Specifc aim 2, and we cannot determine if nNOS is the key isoform re sponsible for atrophy signaling in vitro. Although iNOS is expressed in low levels in skeletal muscle myotubes, it is unlikely that iNOS is active at such low strain. From our results, we remain speculative that nNOS is responsible for the adaptations seen in Akt activation and NFB signaling during our in vitro model of disuse atrophy. Cellular Signaling during High Strain Specific aim 3 tested if excessive load a pplied to skeletal muscle myotubes causes production of high amounts of NO via iNOS and activation of proteoly sis. Skeletal muscle injury leads to inflammation and upregulation of i NOS (140, 147), which is associated with the classical pathway of NFB (I B/p50/p65) (102) to induce pr otein degradation (11). We examined the translocation or degradation of NFB family members as well as iNOS mRNA that are associated with inflammation and injury in the classical pathway of atrophy. We isolated nuclear and cytosolic fractions to probe for the nuc lear localization of the NFB members, p50, p65, Bcl-3, and cytosolic content of I Band MAFbx. MAFbx was measured as

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64 an indirect measure ubiquitin-proteasome activity and protein degradation. Lastly, we used a reporter plasmid to measure NFB-dependent transcrip tional activity as a function of time at high magnitude cyclic strain. Measurements in C2C12 Cell Culture during High Strain C2C12 myotubes were subjected to a high magnit ude stretch (18%; 0.1 Hz) for 1, 2, or 3 h. One group remained inactive for 2 h following the stretch protocol (2hC ES). These time points were chosen to test for a time course of up/downregulation of iNOS mRNA and NFB activity previously shown in other ce ll types (5). The non-specific NOS inhibitor L-NAME was also administered to subcultures of each group throughout the entire protocol. Classical NFB pathw ay The classical NFB pathway involves the nuclear tr ansport of p65/p50 heterodimers by degradation of I Btriggered by its phosphorylation by IKK NO production by iNOS has been shown to induce this classic pathway ( 5, 12). We hypothesized that high magnitudes of stretch induce large amounts of NO (via iNOS) and lead to activation of NFB through the classical pathway. Nuclear p50 Western blot of C2C12 myotubes subjected to high strain revealed that nuclear translocation of p50 significantly peaked after 1 h of 18% stretch (Figure 4-20), and then drifted back toward baseline with continued stretch. Treatment of myotubes with L-NAME was only effective in attenuating nuc lear p50 at the 2 h time point. Nuclear Bcl-3. In accord with the accepted cachexia model of atrophy, we were not able to detect Bcl-3 protein in the nucle ar fractions of myotubes subjec ted to 18% stretch for 3 h. The classical pathway suggests that the p50/p65 heter odimer is sufficient to induce transcription of

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65 atrophy related genes, and that this pathway doe s not involve Bcl-3. Thus high cyclic strain appears to activate the classical pathway rather than the alterna tive pathway of atrophy signaling. Nuclear and cytosolic p65 The localization of p65 tends to be primarily cytosolic as its nuclear transport sequence is blocked by its inhibitor I B. Following I Bphosphorylation and degradation, p65 is free to tran slocate to the nucleus. In orde r to follow this progression we attempted to measure nuclea r p65 and cytosolic p65 and I Bprotein content. Cytosolic levels of p65 did not vary considerab ly throughout the protocol (Fi gure 4-21B). However, nuclear amounts of p65 significantly increased with 1 an d 2 h of stretch (Figure 4-21A and C). L-NAME was overall ineffective at pr eventing p65 nuclear transport. Cytosolic I B. Although statistical significance was not reached, there was a trend for a reduction in cytosolic I Bprotein content during the progre ssion of stretch (Figure 4-22). However, administration of L-NAME to the culture medium increased the loss of I Bprotein after 2 and 3 h of stretch suggesting that NO may actually inhibit phosphorylation and degradation of I Bduring high strain S inhibition demonstrated this same effect on I Bexpression during moderate stretch (Figure 4-18) suggesting that NO may be protective against I Bphosphorylation independent of stre tch magnitude and NO concentration. MAFbx Protein degradation by the 26S proteasom e is dependent on their ubiquitination by E3 ligases. MAFbx is one such muscle-specifi c ligase that has been deemed essential for skeletal muscle atrophy. It is also thought to be involve d in the ubiquitination of I B. MAFbx protein expression increased in a time-depende nt manner through 3 h of 18% cyclic strain (Figure 4-23). Two hours of inactiv ity was sufficient to return MA Fbx levels to baseline. Most interestingly, L-NAME treatment completely abr ogated the increase in MAFbx at all time points

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66 measured indicating that the nitric oxide produced during high strain is necessary for protein degradation in C2C12 myotubes. Inducible NOS gene expression During cach exia and injury, micromolar concentr ations of NO are thought to be produced by iNOS leading to further injury and inflam mation. We sought to determine if iNOS was activated in our model of inflammati on/injury during high cyclic strain. C2C12 myotubes were stretched for 2, 3, or 4 h with one group resting for 2 h following stretch (2hCES). L-NAME was administered in parallel at each time point. Although not significan tly different, there was a trend for an increase in iNOS mRNA expression w ith stretch (Figure 4-24). L-NAME significantly blunted the upregulation of iNOS at every time point. This suggest s that iNOS gene expression is regulated by a positive feedback loop such that inhibition of NO production can prevent transcription of the iNOS gene. Nuclear factor of -B transcriptional activity during high strain To determine if high-magnitude cyclic strain results in NFB-dependent transcriptional activity, C2C12 myotubes were transfected with either a reporter plasmid containing the firefly luciferase gene driven by a promot er sequence containing consensus NFB binding sites (pNFB-luc) or a negative control plasmid (pCIS-CK). No significant differences were detected with any treatment most likely due to our small sample size (Figure 4-25). However, there was a trend for an increase in NFB activity through 2 h of hi gh strain that declined back to baseline over time. In addition, L-NAME treatmen t attenuated any rise in NFB-dependent transcription after 1 h. The trends demonstrated by the NFB reporter plasmid at 18% stretch follow the pattern of both the p65 and p50 subunit translocation to the nucleus (Figures 4-20 and 4-21), which

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67 suggests that the p65/p50 heterodimer is most like ly involved in gene regulation using our model of injury. Inducible NOS Knockout Prim ary Satellite Cell Culture To test which isoform of NOS is involved in the stretch-induced cha nges in the classical pathway of NFB signaling, we isolated satellite cells from mice null for the iNOS gene. After differentiation, the primary myotubes were stre tched for 3 h followed by 2 h of cessation of stretch in accordance with the high strain protocol used for the C2C12s. Subgroups were also treated with L-NAME in an effort to distinguish the effects of iNOS from that of eNOS and nNOS. Visually, the myogenic ce lls isolated from the iNOS-/mice showed enhanced proliferation rates in compar ison to wildtype myoblasts; how ever, both cultures achieved full differentiation after 5 d. After implementing the 18% cyclic stretch pr otocol, myotubes from the wildtype mice demonstrated very similar results for NFB protein expression as seen in the C2C12 myotubes (Figures 4-20 through 4-22). The cytosolic proteins, p65 and I B, did not change in wildtype or knockout myotubes (Figures 427B and 4-28); however, nuclear levels of p65 and p50 varied considerably with treatment. Wildtype myot ubes exposed to stretch experienced a timedependent increase in nuclear p50 which was a ttenuated with NOS inhibition (Figure 4-26). Inducible NOS knockout myotubes had a similar re sponse to stretch alth ough the increase in p50 was significantly reduced. Further, L-NAME co mpletely abrogated p50 protein expression in iNOS-/myotubes at all time points (Figure 4-26) Wildtype and iNOS null mice both showed a progressive increase in nuclea r p65 with stretch over 2 h (Figur e 4-27A and C), and once again, L-NAME completely prevented any increase of nuclear p65 in iNOS-/myotubes during stretch.

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68 We also attempted to measure MAFbx and Bcl-3 expression in both wildtype and iNOS knockout myotubes. However, neither of these pr oteins could be dete cted by western blot. Altogether, these results demonstrate that 18% cyclic strain is sufficient to induce iNOS production of NO which activates the classical pathway of NFB signaling. Concomitant administration of L-NAME to iNOS(-/-) myotubes further inhibited the pathway suggesting that either nNOS or eNOS may also contribute to the production of nitric oxide involved in this signal.

PAGE 69

69 Figure 4-1. Representative images of C2C12 myotubes using the atrophy protocol. Top row indicates cells cultured for 2 d with no stretch (2dCON) and with L-NAME (2dLN). Second row shows cells exposed to 2 d of 12% cyclic strain for 1 h/d (2dSTR) in the presence of L-NAME (2dSTR+LN). Third row represents cells af ter 2 d of stretch and 3 subsequent days of no stretch (2dSTR3dCES) and with L-NAME in the medium (2dSTR3dCES+LN). Fourth row de notes myotubes after 5 d of no stretch (5dCON) and with L-NAME (5dLN). Bottom ro w indicates cells subjected to 5 d of continuous stretch (1 h/d) (5dSTR) in the presence of L-NAME (5dSTR+LN).

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70 Figure 4-1 Continued.

PAGE 71

71 Figure 4-2. Image analysis of C2C12 myotubes using the atrophy pr otocol. A) Average myotube length. B) Average myotube diameter. C) Average myotube area. Values represent the mean SEM. (n=35) *Significantly different from 2dCON. # Significantly different from 2dSTR. f Significantly different from 5dCON. Significantly different from 2dSTR3dCES. (p<0.05)

PAGE 72

72 Figure 4-3. AlphaII-spectrin protein degradation of C2C12 myotubes using the atrophy protocol. Graphs represent ratio of cleaved to intact II-spectrin protein. A) Calpain-specific 150 kDa II-spectrin cleavage product. B) Calpain-specific 145 kDa II-spectrin cleavage product. C) Ca spase-3-specific 120 kDa II-spectrin cleavage product. Values represent the mean SEM. (n=5) *Significantly different from 2dCON. # Significantly different from 2dSTR. f Significantly different from 5dCON. Significantly different from 2dSTR3dCES. (p<0.05)

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73 Figure 4-4. Talin protein degradation of C2C12 myotubes using the atro phy protocol. Graphs represent ratio of the 190-k Da cleavage product to the 235kDa intact talin protein. Values represent the mean SEM. (n=3) *Significantly different from 2dCON. # Significantly different from 2dSTR. Significantly different from 2dSTR3dCES. (p<0.05)

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74 Figure 4-5. Integrin 1 protein expression in C2C12 myotubes using the at rophy protocol. Graph represents integrin 1 normalized to -actin. Values represent the mean SEM. (n=4) *Significantly different from 2dCON. # Significantly different from 2dSTR. f Significantly different from 5dCON. Significantly differe nt from 2dSTR3dCES. (p<0.05)

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75 Figure 4-6. Total pr otein content of C2C12 myotubes using the atrophy protocol. Graph represents total protein cont ent in whole cell lysate. Va lues represent the mean SEM. (n=3) Significantly different from 5dCON. (p<0.05)

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76 A B Figure 4-7. Representative images of C2C12 myotubes immunostained for dystrophin and MHC type IIa and corresponding negative controls after 6 d of differentiation. A) Immunostain of dystrophin and myosin heavy chain type IIa. B) Negative control for MHCIIa (FITC) and dystrophin (Rhodamine).

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77 Figure 4-8. Representative images of C2C12 myotubes immunostained for nNOS, iNOS, and eNOS after 6 d of differentiation.

PAGE 78

78 Figure 4-9. Representative images of C2C12 myotubes immunostained for nNOS using the atrophy protocol.

PAGE 79

79 Figure 4-9 Continued.

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80 Figure 4-10. Total nitrate plus nitrite concentrations throughout the atrophy protocol. Culture medium was collected during th e final 48 h of treatment. Values represent the mean SEM. (n=6) Significantly different from 5dSTR. (p<0.05)

PAGE 81

81 Figure 4-11. Ratio of phosphorylated to total Akt protein expression in C2C12 myotubes 12, 24, and 48 h after 12% stretch. Values represent the mean SEM. (n=3) Significantly different from baseline. # Significantly differe nt from Control. f Significantly different from Stretched Control. Significant main effect of time, L-NAME, and interaction. (p<0.05)

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82 Figure 4-12. Ratio of phosphorylated to total GSK-3 protein expression in C2C12 myotubes 12, 24, and 48 h after 12% stretch. Values represent the mean SEM. (n=3) Significantly different from baseline. # Significantly different from Control. Significant main effect of L-NAME and interaction. (p<0.05)

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83 Figure 4-13. Phosphorylated F OXO3a protein levels in C2C12 myotubes 12, 24, and 48 h after 12% stretch. Values represent the mean SEM. (n=3) Significantly different from baseline. # Significantly different from Control. f Significantly different from Stretched Control. Significant main eff ect of time, L-NAME, and interaction. (p<0.05)

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84 Figure 4-14. Muscle atrophy F-box (MAFbx) protein expression in C2C12 myotubes 12, 24, and 48 h after 12% stretch. Values represent the mean SEM. (n=3) Significantly different from baseline. Significant main effect of time. (p<0.05)

PAGE 85

85 Figure 4-15. Nuclear p50 protein levels in C2C12 myotubes 12, 24, and 48 h after 12% stretch. Values represent the mean SEM. (n=3) Significantly different from baseline. # Significantly different from Control. f Significantly different from Stretched Control. Significant main effect of time and interaction. (p<0.05)

PAGE 86

86 Figure 4-16. Nuclear Bcl3 protein expression in C2C12 myotubes 12, 24, and 48 h after 12% stretch. A) Ratio of phosphorylated to total Bcl-3 protein. (Significant main effect of L-NAME and interaction.) B) Total Bcl-3 prot ein. (Significant main effect of time.) Values represent the mean SEM. (n=3) Significantly different from baseline. # Significantly different from Control. f Significantly different from Stretched Control. Significantly different from Stretched L-NAME. (p<0.05)

PAGE 87

87 Figure 4-17. Nuclear and cytoso lic p65 protein levels in C2C12 myotubes 12, 24, and 48 h after 12% stretch. A) Nuclear p65 le vels. (Significant main effect of drug.) B) Cytosolic p65 levels. (Significant main effect of dr ug.) C) Ratio of nuclear to cytosolic p65. (Significant main effect of drug, time, and interaction.) Va lues represent the mean SEM. (n=3) Significantly different from baseline. # Significantly different from Control. f Significantly different fr om Stretched Control. Significantly different from Stretched L-NAME. (p<0.05)

PAGE 88

88 Figure 4-17 Continued.

PAGE 89

89 Figure 4-18. Cytosolic I Bprotein expression in C2C12 myotubes 12, 24, and 48 h after 12% stretch. Values represent the mean SEM. (n=3) Significantly different from baseline. # Significantly different from Control. f Significantly different from Stretched Control. Significantly different from Stretched L-NAME. Significant main effect of time, dru g, and interaction. (p<0.05)

PAGE 90

90 Figure 4-19. Nuclear factor of B (NFB) transcriptional activity in C2C12 myotubes 12, 24, and 48 h after 12% stretch. Graph represents NFB-dependent transcriptional activity (NFB reporter plasmid indicated by pNFB-luc) or empty vector activity (pCIS-CK) relative to uptake control (pRL -CMV). Values represent the mean SEM. (n=3) No statistical differences.

PAGE 91

91 Figure 4-20. Nuclear p50 protein expression in C2C12 myotubes subjected to high cyclic strain for 3 h. Values represent the mean SEM. (n=3) Significantly different from baseline. # Significantly different fr om Stretch. Significant main effect of time and LNAME. (p<0.05)

PAGE 92

92 Figure 4-21. Nuclear and cytoso lic p65 protein expression in C2C12 myotubes subjected to high cyclic strain for 3 h. A) Nuclear p65 leve ls. (Significant main effect of L-NAME, time, and interaction). B) Cytosolic p65 levels. (Significant main effect of time.) C) Ratio of nuclear to cytosolic p65. (Signi ficant main effect of L-NAME and time.) Values represent the mean SEM. (n=3) Significantly different from baseline. # Significantly different from Stretch. f Significantly different from 2hSTR. Significantly different from 1hSTR. (p<0.05)

PAGE 93

93 Figure 4-21 Continued.

PAGE 94

94 Figure 4-22. Cytosolic I Bprotein expression in C2C12 myotubes subjected to high cyclic strain for 3 h. Values repres ent the mean SEM. (n=3) Significantly different from baseline. Significant main effect of time. (p<0.05)

PAGE 95

95 Figure 4-23. Muscle atrophy F-box (MAFbx) protein expression in C2C12 myotubes subjected to high cyclic strain for 3 h. Values represent the mean SEM. (n=3) Significantly different from baseline. # Significantly different from Stretch. f Significantly different from 3hSTR. Significant main effect of L-NAME, time, and interaction. (p<0.05)

PAGE 96

96 Figure 4-24. Inducible NOS (iNOS) mRNA levels in C2C12 myotubes during 4 h of high cyclic strain. Values represent the mean SEM. (n=3) Significant main effect of L-NAME treatment. (p<0.05)

PAGE 97

97 Figure 4-25. Nuclear factor of B (NFB) transcriptional activity in C2C12 myotubes subjected to high cyclic strain for 3 h. Graph represents NFB-dependent transcriptional activity (NFB reporter plasmid indicated by pNFB-luc) or empty vector activity (pCIS-CK) relative to uptake control (pRL -CMV). Values represent the mean SEM. (n=3) No statistical differences.

PAGE 98

98 Figure 4-26. Nuclear p50 protein expressi on in primary cultured myotubes from iNOS-/and wildtype mice subjected to high cyclic strain for 3 h. Values represent the mean SEM. (n=3) Significantly different from baseline. # Significantly different from Stretch. f Significantly different from previous hour treatment. Significantly different from Wildtype. Significant main effect of L-NAME, time, and interaction. (p<0.05)

PAGE 99

99 Figure 4-27. Nuclear and cytosolic p65 protein expression in primary cultured myotubes from iNOS-/and wildtype mice subjected to high cy clic strain for 3 h. A) Nuclear p65 levels. (Significant main effect of L-NAME, time, and interaction.) B) Cytosolic p65 levels. (Significant main effect of L-NAM E, time, and interaction.) C) Ratio of nuclear to cytosolic p65. (Significant main effect of time and interaction.) Values represent the mean SEM. (n=3) Significantly different from baseline. # Significantly different from Stretch. f Significantly different from previous hour treatment. Significantly different from Wildtype. (p<0.05)

PAGE 100

100 Figure 4-27 Continued.

PAGE 101

101 Figure 4-28. Cytosolic I Bprotein expression in primary cultured myotubes from iNOS-/and wildtype mice subjected to high cyclic strain for 3 h. Values represent the mean SEM. (n=3) No statistical differences.

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102 CHAPTER 5 DISCUSSION Main Findings This is the first study to our knowledge to de velop two com pletely intrinsic models of skeletal muscle atrophy in vitro : 1) withdrawal from moderate cyclic stretch, and 2) high magnitude cyclic strain. First, moderate cyclic mechanical stretch can be used as a model of activity in cultured skeletal muscle myotube s, and increases myotube size through NOSdependent induction of Akt signali ng. Cessation of this type of moderate stretch causes protein degradation, altered nNOS loca lization, and a reduction in myot ube size via downr egulation of Akt, which may contribute to NFB signaling through an alternative pathway. Secondly, high magnitude cyclic strain can indu ce the classical pathway of NFB signaling and upregulate iNOS in cultured skeletal muscle myotubes. These data demonstrate in vitro models of atrophy independent of external factors and provide evidence to better understand the signaling pathways involved during skeletal muscle loss. Disuse Atrophy Model Cessation of Cyclic Stretch Causes Sk eletal Muscle Myotube Atrophy In vivo skeletal muscle atrophy is defined as a loss in skeletal muscle mass, and can be measured as a reduction in fiber cross-sectiona l area and protein content, reduced force and power output, increased fatigability and increased insulin resistance. We define skeletal muscle myotube atrophy as a decrease in myotube size, increased protein degradation and protein loss. Cyclic mechanical stretch in culture elicits the acute effects similar to muscle activity in vivo such as membrane deformation (28), increased glucose uptake (86), protein synthesis (81), and free radical production (102), as well as some of the chronic adapta tions associated with exercise, including myotube hypertrophy (188), increased cytoskeletal stability (212), and

PAGE 103

103 reduced fast myosin heavy chain composition (122). Thus, cyclic mechanical stretch to skeletal muscle myotubes can be used as a denervated model of skeletal muscle activity in vitro In our model, we demonstrated that five days of a 12% cyclic stretch applied to C2C12 myotubes was sufficient to cause myotube hypertrophy through an increase in myotube diameter and area (Figures 4-1 and 4-2) although no change in to tal protein content was evident (Figure 4-6). Passive stretch of mature musc le leads to an increase in muscle tension which plays an important role in cell prolifer ation, differentiation, metabolism, remodeling, and survival (30, 46, 94, 104, 143). Many of these processes are dependent on activation of integrin-mediated signaling pathways (93, 124, 156). This notion is supported by an in vivo study that showed impaired fusion and defective cytoskeleton in 1D-integrin-deficient myobl asts (158). Thus, it is conceivable that the expression of 1-integrin is required to strengthen the linkage between cytoskeleton and extracellular matrix in the developing myotube. Localization of 1-integrin at the sarcolemma in skeletal myotubes may enable it optimally to sense changes in the mechanical environment, where it serves an important role in sensing and transduc ing mechanical signals from the external environment to the cytoplasm ( 14). In agreement with the literature (213), we showed that multiple days of 1-h bouts of cyc lic stretch increased the mechanotransduction signaling protein 1-integrin in C2C12 myotubes (Figure 4-5). Beta1-integrin may also interact with Rho GTPases, and the activity of Rho GTPases is critical for integrin clustering and partial phosph orylation of FAK (35, 37, 54). Similar to FAK, Rho GTPases play an important role in myodi fferentiation. These Rho fa mily proteins also regulate the organization of th e actin cytoskeleton. In C2C12s, a 10% cyclic stretch successfully activated RhoA, GTP-binding proteins, FAK, and Z-line formation (36, 213), as well as RhoA

PAGE 104

104 protein expression (120). Thus, repetitive stretch of myotubes in vitro can initiate cytoskeletal formation and mechanotranduction pathways as sociated with skeletal muscle activity. On the other hand, inactivity of skeletal muscle causes fiber atrophy. Removal of mechanical stretch caused myotubes to respond as if they had been forced to an inactive state. Here, we demonstrated that C2C12 myotubes experience cytoskeletal protein degradation (Figures 4-3 and 4-4) and a decrease in size after three days of no stretch (Figures 4-1 and 4-2). Thus, forced inactivity of skeletal muscle myotubes through cessation of daily repetitive stretch is sufficient to induce visible and measurable myotube atrophy. Skeletal muscle atrophy is predominately due to increased proteolysis leading to a loss of mass. Evidence indicates that the rate-limiting step in protein degradation during skeletal muscle atrophy is the actomyosin disassociation by calpain and/or caspase-3. The cl eavage products of two cytoskeletal proteins, II-spectrin and talin, that are known to be substrates of calpain and caspase were increased during the inactivity peri od of our protocol (Fig ures 4-3 and 4-4). Evidence that ubiquitous calpains are key players in atrophy is based on expression analysis, detection of activity of calpain and the use of calpain inhibitors. Several studies have established that calpains are elevated in atrophic conditions lik e disuse, denervation, glucocorticoid treatment and sepsis (72, 79, 172, 192, 199). Calpain substrates include proteins that are important to sarcomeric structural integrit y, such as Z-disk proteins myofibrillar proteins and structural cytoskeletal protei ns (65, 198) including, talin, vinculin, II-spectrin, nebulin and titin (82, 116). Ca2+ spikes, removal of the N-terminus region by autolysis, calpastatin and nitric oxide, all seem to be involved in the regula tion of ubiquitous calpain activity (66, 123). Interestingly, the inhibition of nitric oxide synthase with L-NAME in C2C12 myotubes induced talin degradation independe nt of stretch (Figure 4-4). N itric oxide has been shown to

PAGE 105

105 inhibit calpain activity via S-nitrosylaton of an active site cysteine, and the NO donor sodium nitroprusside was sufficient to prevent vinculin and talin degradation after calcium ionophoretreatment in C2C12 myoblasts (99). Thus, endogenous nitr ic oxide production may be protective against calpain-induced proteolysis. Since nitric oxide production is enhanced by cyclic stretch (Figure 3-3), this may partly explain why cytoskeletal proteo lysis by calpain was reduced by mechanical activity in our model. Localization and Activity of NOS after Stretch Skeletal m uscle contains all three isofor ms of nitric oxide synthase, neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS), and the localization of each NOS isoform in skeletal muscle va ries (Figure 4-8) (18, 148). Endothelial NOS is mainly expr essed in the cytoplas m of all fibers, but mostly types I and IIa (76), where it is colocalized with mito chondrial markers within rat diaphragm, EDL, gastrocnemius, and soleus muscles (98). Immunostaining of eNOS in C2C12s demonstrated its diffuse location throughout the myotubes (Figur e 4-8). We cannot ascertain if eNOS is associated with mitochondria in these cells or merely maintained a cytosolic residence. Inducible NOS is present at very low levels in healthy skeletal muscle of humans (135) and rodents (177) but is enhanced in normal C2C12 myotubes (18) and in resp onse to endotoxin (177). The presence of iNOS is normally cytosolic or co ncentrated at the neurom uscular junction (206); however, our C2C12 immunohistochemical analyses showed s potty staining similar to that of nNOS (Figure 4-8) suggesting a membrane-bound locality. Inducible NOS is constitutively expressed in skeletal muscle fibers of pathogen-free guinea pigs (61) where it copurified with a pooled particulate protein fraction and showed a spotty in tracellular appearance in type I fibers of the diaphragm and gastrocnemius muscles (61). Others reported that iNOS coimmunoprecipates with the sarcolemmal caveolae membra ne protein, caveolin-3, thus providing a molecular basis

PAGE 106

106 for a sarcolemmal localization of iNOS (62). Further, in muscle fibers of patients with autoimmune inflammatory myopathies, an intense immunocytochemical labeling was observed along large stretches of the sarcolemma, in contrast to small spots seen in normal muscle (174). In human vastus lateralis and soleus muscle, iNOS is associated with subsarcolemmal caveolin-3 primarily in Type I fibers; however, after 90 d of bed rest, iNOS is no l onger detectable at the membrane is seen only in the sarcoplasm (148). All together, the eviden ce suggests that in healthy tissue iNOS may be localized at the membrane in relatively low levels in most mammals and more highly expressed in C2C12 myotubes and guinea pigs. Ho wever, iNOS localization differs in response to unloading, endotoxin, or disease where it may become upregulated and maintained throughout the sarcoplasm. Neuronal NOS is associated with the dystroph in-glycoprotein complex which determines its sarcolemmal positioning in normal adult myofiber s (4). This prominent molecular association of nNOS to the sarcolemmal DGC is reflected by the ring-like immunos taining patterns (60, 69). We also witnessed this ring-like appearance in C2C12 myotubes after 6 d of differentiation (Figure 4-8) and was enhanced after repeated bouts of cyclic st retch (Figure 4-9). Disruption of the NOS dystrophin complex in mdx mice and human Duchenne musclular dystrophy results in displacement of NOS protein from the sarcolemma to the cytosolic compartment in myofibers (23). Also, in human atropic and necr otic muscle fibers, nNOS displayed an enhanced cytoplasmic nNOS distribution (174). More recen tly, Suzuki and colleagues demonstrated nNOS becomes dislocated from DGC during hindlimb unloading and becomes cytoplasmic where it facilitates activation of FOXO3a, MuRF-1, and MA Fbx leading to skeletal muscle atrophy (169). Athough we did not co-stain for nNOS and dystrophin, we did demonstrate that C2C12 myotubes are capable of expressi ng dystrophin after 6 d of differ entiation (Figure 4-7A). The

PAGE 107

107 literature endorses the association of nNOS and the DGC as well as its dissociation during disuse and disease. Thus the lack of spottiness in myot ubes subjected to a 3 d withdrawal of stretch is likely due to nNOS dissociation from the DGC in response to atrophic cy toskeletal degradation and release of structural protei ns located at the sarcolemma. Long term (5 days) exposure to L-NAME was also capable of causing sarcolemmal dislocation and nNOS diffusi on throughout the cytoplasm (Figure 4-8). L-NAME has been shown to inhibit increased nNOS protein expression after stretch as well as levels of desmin, vinculin and talin in C2C12 myotubes (212) Further, nNOS overexpression is sufficient to increase mRNA and protein levels of integrin, vinculin, talin, dystr ophin, dystroglycans, and syntrophins (180). Renormalizati on of NO production in mdx muscle was sufficient to reduce membrane damage (197) possibly due to increased e xpression of structural proteins at the cell membrane (51) such as talin (179) vinculin (179), and utrophin (34). Altogether, it appears that nitric oxide production is essential for expression and maintenance of cytoskeletal and DGCassociated proteins. Inhibition of NOS with L-NAME may cause the cytoskeleton to be more susceptible to calpain-stimulated degradation (99) thereby releasing nNOS into the cytoplasm. NOS activity did not significant change during our disuse atrophy model; however levels of NO production did highly correlate with stretch and 1-integrin protein concentrations (Figures 4-10 and 4-5). Genetic knockout of the 1-integrin gene completely eliminated NO production following 10% stretch in C2C12s, but 1-integrin reincorporation into the muscle membrane restored normal NO levels (213). This suggests that n NOS association with 1integrin is necessary for the stretched-induced NO S activity seen in our model. Loss of integrin and its membrane-associated proteins during atrophy may disrupt mechanotransduction pathways and influence NO production during atrophy or other pathologies.

PAGE 108

108 Stretch-Induced Activation of Akt is NOS-Dependent The m echanisms by which growth factors, su ch as insulin and IGF, simulate protein synthesis in skeletal muscles have been well characterized. This includes activation of Akt, which occurs through a PI3K-dependent mechanis m (96). In turn, activated Akt phosphorylates GSK-3 resulting in an inhibition of GSK-3 activity (152, 187). The decrease in GSK-3 activity is linked to a de crease in phosphorylation of eIF2B at Ser535, an ev ent that is linked to enhanced eIF2B activity and prot ein synthesis (141, 195). Insulin also induces an increase in p70S6k phosphorylation through a PI3K and mTOR-dependent pathway (29, 44, 85). Activated p70S6k is known to phosphorylate the S6 subunit of the 40 S ribosome, an event that has been implicated in the translationa l control of RNAs (88, 95). Thus after insulin/IGF1 stimulation, both GSK-3 and p70S6k contribute to the PI3Kand mTOR-d ependent pathways that regulate protein synthesis. Similar to insulin, mechanically-induced increas es in protein synthesis were found to be both PI3Kand mTOR-dependent, and highly correlated with the changes in GSK-3 and p70S6k phosphorylation (81). However, in both the co -incubation and conditione d-media experiments, the release of locally acting factors was not su fficient for the activation of mTOR-dependent signaling events, thus suggesti ng that mechanotransduction (e.g., mechanoreceptor) rather than ligand binding of autocrine/paracrine growth fact ors as the cause for the induction of the mTORdependent signaling events (81). The PI3K-Akt signaling pathway is now one of the most recognized mechanosensitive signaling pathways in skeletal muscles. Several reports have convincingly shown the activation of Akt by increased skeletal muscle contraction or passive muscle stretch (81, 146, 151, 152, 185). Akt signaling contributes to muscle mass growth and maintenance by two independent mechanisms: one involves stimulation of protein synthesis (as previously discussed), and the

PAGE 109

109 other includes inhibition of protein degradation. Akt inhi bition of protein degradation acts through downregulation of the family of FOXO tran scription factors, whic h are responsible for expression of the muscle atrophy-induced ubiquitin ligases MuRF1 and MAFbx (154, 166). Regulation of FOXO factor activity is mainly controlled by a shuttling system that modulates its cellular localization by phosphorylation sites located in the COOH-terminal domain. Phosphorylation of these sites by Akt provokes th eir nuclear export (186). Four members of the FOXO subfamily of fo rkhead transcription factors have been identified in the mouse; and in C2C12 myotubes, the expression of constitutively active Foxo3a is enough to induce atrophy, with a concomitant activation of MAFbx expression (154). Further, passive stretch of the diaphragm was sufficien t to induce PI3K-Akt and inhibited DNA binding of FOXO1 and FOXO3a (134), sugg esting that a stretch stimulus is sufficient to suppress the signals leading to protein degradation. In agreement with the literature, our m odel of cyclic mechanical stretch in C2C12 myotubes induced a large and transient increase in Akt activity, which was complimented by phosphorylation of GSK-3 and inhibition of FOXO3a (Fi gures 4-11 through 4-13). Thus, membrane deformation via mechanical stre tch of myotubes is sufficient to activate mechanosensitive pathways necessary to enhance protein synthesis and prevent proteolysis. Likewise, cessation of mechanical stretch fo r 12-48 h caused a significant reduction in Akt activity and a concomitant increase in FOXO3a (Figures 4-11 and 413). However, this length of time was not sufficient to induce significant MA Fbx expression. In spite of this, protein degradation and myotube atrophy did occur within 72 h withdraw al of stretch (Figures 4-2 through 4-4), and we can speculate that perhaps the other muscle -specific E3-ligase MuRF1, and not MAFbx, was involved in taggi ng proteins for degradation in our model. Others have shown

PAGE 110

110 MuRF1 to be associated with sarcomeric proteins in skeletal muscle (121), and thus MuRF1 may play a larger role in myofibrillar ubiq uitination during myotube atrophy than MAFbx. Most interestingly, the Akt res ponse to stretch was completely dependent on nitric oxide synthase activity, as L-NAME blocke d both phosphorylation of Akt and GSK-3 and led to increased activity of FOXO3a. Our lab was the first to demonstrate the ability of nitric oxide to augment Akt activity in skeletal muscle cells Low doses of the NO donors SNAP and PAPANO (1 and 10 M) were sufficient to i nduce phosphorylation of Akt; whereas, higher concentrations (100-1000 M) inhibited Ak t phosphorylation (48), presumably via Snitrosylation of cysteine 224 independent of PI3K signaling (207). Thus, the nitric oxide regulation of Akt is dose-dependent and may be a ltered during various magn itudes of strain (80). The mechanism for how NO facilita tes activation Akt is unknown, but it is thought that NO may influence upstream targets such as PI3K or protein phosphatases (48). Moderate Stretch and Pathways of NFB Signaling The NFB family of transcription factors is well documented as being involved in both cachexia and disuse forms of skeletal muscle atrophy. However, NFB has also been shown to increase with exercise and st retch. Our data shows that NFB-dependent transcription and the NFB subunits p50, Bcl-3 and p65 become temporarily elevated in the nucleus following repetitive bouts of 12% cyclic strain in C2C12 myotubes (Figures 4-15 through 4-17, 4-19). Acute bouts of treadmill exercise, muscle contracti on, and passive stretch transiently induce I B kinaseand (IKK / ) and I B phosphorylation, nuclear import of p65/p50, and enhanced NFB binding that peaks within 2 h of exerci se in mice (77, 102, 103) and rats (89). The elevated NFB activity following stretch was prevente d by the antioxidant NAC (103), and the exercise-induced NFB binding to DNA was asso ciated with binding domains of antioxidant genes (i.e., MnSOD) and was accompanied by an elevation of endogenous antioxidant enzyme

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111 RNA and protein content (78). Thus, NFB activation may be stimulated by ROS production following exercise or stretch and may be an underlying mechanism for training adaptations and increased expression of antioxidant enzy mes. Further, the upregulation of NFB binding following exercise training stimulates cellular pr otection against apoptosi s and reduction of the pro-apoptotic proteins (1, 64, 90). The induction of NF-kB subunits p50/p65 immediately after stretch in our model is likely due to phosphorylation of IKK by Akt. Of the se veral kinases known to activate IKK, Akt is the most prominent (173, 181), and we show that Akt phosphorylation increases ~5 fold after stretch (Figure 4-11). IKK activation leads to subsequent phosphorylation of the NFB inhibitory protein I B causing its ubiquitination and degradation by the proteasome. This results in translocation of NFB dimers to the nucleus and initiation of gene expression (105). Mechanical stretch has been shown to activate NFB transcription factors in myofibers through a PI3K/Aktdependent pathway. Inhibition of PI3K and Akt abrogated IKK activity and prevented degradation of the inhibitory subunit I B (47). Thus, this suggests that activation of NFB transcription may be due to increased IKK activity by phosphorylation of Akt. Although the p65/p50 heterodimer release by I B degradation follows the classical NFB pathway induced with stress, we cannot rule out the possibility of an alternative pathway, which involves the DNA binding of p50 homodimers in combination with Bcl-3, since nuclear levels of all three subunits were elevated in our model of stretch (Fi gures 4-15 through 4-17). The Akt-induced IKK activation has also been shown to phosphorylate p105, which is the inhibitory subunit of p50 (153). Phosphorylation of p105 involves ubiquitination and subsequent degradation by the 26 S proteasome (132). However, a glycine-rich region in the C-terminal half of the p50 moiety acts as a physical barrier to the proteasome and causes limited proteolysis

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112 (117, 130). This process reveals a nuclear transl ocation sequence allowing p50 to bind to DNA. However, p50 lacks a transactivat ion domain and cannot initiate gene transcription on its own. The I B family member Bcl-3 has a high a ffinitiy for p50 homodimers, and p50/Bcl-3 complexes can activate transcript ion through the Bcl-3 transactiv ation domain (22, 57, 133). This agrees with our model in C2C12 myotubes which showed a 4-fold increase in both nuclear p50 and Bcl-3 after a 1-h bout of cyclic stretch (Figure 4-15 and 4-16B). Another link between Akt signaling a nd the alternative pathway of NFB signaling may contribute to myotube atrophy dur ing cessation of stretch. Along with the decrease in Akt activity 12-48 h post-stretch, we sa w a concomitant increase in GSK-3 activity (Figures 4-11 and 4-12). GSK-3 is a kinase known to phosphorylate Bcl-3, and the NFB p50/Bcl-3 complex becomes more transcriptionally active when Bcl-3 is phosphory lated (25, 129). The increased ratio of phosphorylated-to-total Bcl-3 followed the same trend as GSK-3 activity (Figure 416A) suggesting that the alternative pathway of NFB is activated during cessation of stretch in our model. Also, similar to Akt, both Bcl-3 and GSK-3 appear to be regulated by nitric oxide production, since the activities of both were significantly augmented with NOS inhibition after stretch. Thus, the skeletal muscle myotube atro phy witnessed during 3 d of stretch withdrawal may be due in part by reduced Akt ac tivity, increased activation of GSK-3 and enhanced phosphorylation of the NFB complex p50/Bcl-3 to induce atrophy related genes. Lastly, the fact that NOS inhibition induced nuclear levels of p65 and NFB-dependent transcriptional activity during cessation of stretch was unexpected (Figures 4-17 and 4-19); however, they may be interconnected. Nitric oxi de has been shown to induce and stabilize I B (137), which would contribute to maintenan ce of p65 in the cytosol. By inhibiting NO

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113 production, I B may be more susceptible to degradation, less abundant, and allow for p65 nuclear translocation to c ontribute to enhanced NFB transcription. Inflammation-Associated Atrophy Model High Magnitude Stretch Induces iNOS and NFB Cachexia and muscle injury are known to cau se a robust increase in various cytokines, iNOS, and NFB pathways which lead to skeletal muscle breakdown and atrophy (56, 147). High strain applied to sk eletal muscle myotubes in vitro is sufficient to induce myotube injury, superoxide production (184) as well as upregulate cytokines and NFB signaling through mechanosenstive pathways (211). Therefore, high magnitude cy clic stretch of C2C12 myotubes is an appropriate model in which to study the intrinsic signaling pa thways involved in cachexia and inflammation-associated atrophy. Mechanotransduction pathways are sensitive to load, velocity and magnitude of the stress imposed upon the muscle, and the signal that is tranduced from the muscle membrane to the interior of the cell will regulate the downstream pa thways (27). Moderate stretch, as discussed in the previous section, can elicit anabolic, adap tive, and protective signal s within skeletal muscle. Conversely, higher magnitudes of stretch may be deleterious to muscle and the subsequent response is to induce mediators of injury a nd inflammation which leads to muscle breakdown and degradation. In general, 10-12% stretch of muscle cells in culture, which causes cell deformation without significant injury, is considered to be a model fo r mechanical activity; whereas stretch of 17-20% or greater is consid ered an injury model (157, 189). Thus, we chose to stretch C2C12 myotubes by 18% as a model fo r inflammation-induced atrophy. The proinflammatory cytokine TNF, a potent activator of NFB in muscle cells (113) plays a pathological role in mediation of such disorders as cachexic muscle wasting, inflammatory myopathies, and insulin resistan ce (119, 144). Although, as a regulator of immune

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114 and inflammatory response, TNFis primarily synthesized by macrophages and other immune cells (183), but it is also synthe sized by skeletal muscle in a hi ghly regulated manner. Myoblasts constitutively synthesize TNF(150), and this activity increases transiently upon differentiation (115). Myofibers respond to various types of injury with drama tically increased expression of TNFand its receptors (38, 42, 175, 196, 210). In addition, strenuous exer cise elevates the level of circulating TNF(131, 165), which originates from skeletal muscle (128, 165). TNFis synthesized as a 26 kDa pro-TNF, which is then anchored to the plasma membrane. The membrane-bound pro-TNFis cleaved and released as a 17 kDa secreted form of TNFby TNFconverting enzyme (TACE). TACE, also known as ADAM17, is a ubiquitous enzyme that belongs to the ADAM family of disintegrin metalloproteinases (16, 17 ) and is key regulator of the availability of autocrine TNFin mechanotransduction (211). Tumor necrosis factoris one of the main activators of the NFB pathway (24, 71, 92), and it is well established that cachexic muscle atrophy requires the activation of transcription factors including NFB (31) and Foxo3a (154), leading either to the rapid decrease of MyoD mRNA (71) or to the ove rexpression of the ubiquitin ligase, MAFbx (154). NFB regulates the expression of a wide variety of genes, includi ng those encoding cytokines, chemokines, adhesion molecules, and inducible enzymes (e.g., iNOS and COX-2) (63). It was previously shown that TNFinduces the expression of iNOS, leading to the production and release of NO and, oxidative stress in the skeletal muscles of cachexic animals and C2C12 myocytes (24, 200). The release of NO by iNOS depends on the transcript ion of the NOS gene. The murine and human iNOS promoters contain several binding site s for transcription factors such as NFB (6). Regulation of iNOS via the NFB pathway is an important mechanism in the inflammatory process and constitutes a poten tial target to combat inflammation-related disease.

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115 In our in vitro model of inflammation-associated atr ophy, we demonstrated that 18% cyclic stretch to C2C12 myotubes significantly increases NFB nuclear translocation and NFBdependent transcription leading to the upregulation of iNOS a nd MAFbx expression (Figures 420 through 4-25). The I B member, Bcl-3, was undetectable in all samples suggesting that the classical pathway of NFB signaling (i.e., I B/p50/p65) was exclusivel y activated in our model. This agrees with the observation that TNF-induced activation of NFB activates the prototypical p65-p50 heterodimer in skeletal muscle (127). The inflammation-associated, TNF-dependent pathway of NFB signaling has been shown to initiate degradation of intrinsic muscle proteins as well as inhibit myogenesis through a complex list of target genes (70). Using dominant negative inhibition of NFB, investigators have shown that TNF-induced NFB activation is responsible for an increase in ubiquitinconjugating activity and upregulation of the ubiquitin-conjugating E2 enzyme, called UbcH2 (114). Another target gene of NFB in muscle cells is the C3 proteasome subunit (49), which again implicates NFBs involvement in the ubiquitin-proteasome pathway of protein degradation. Additiona lly, treatment of C2C12 cells with TNF, leads to the lost expression of MyoD, myogenin, CDK, myosin heavy chain, and tropomyosin proteins, resulting in the eventual inhibition of myot ube formation (3, 70). TNFinhibits skeletal muscle differentiation via the induction of iNOS and NO production in a NFB dependent manner, which subsequently leads to MyoD mRNA degenera tion (45, 71). Furthermore, several genetic approaches have demonstrated that TNFand its downstream effectors negatively regulate the process of myogenesis in vitro (71, 106). Therefore, inflam mation-associated atrophy through NFB and iNOS is likely due to downregulation of myogenesis and incr eased proteolysis of skeletal muscle cells.

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116 Nitric Oxide Synthase I nhibition Prevents NFB Nuclear Translocation, iNOS and MAFbx Expression during High Strain The inhibition of NOS during 1-3 h of 18% cyclic mechanical strain in C2C12 myotubes significantly abrogated the degradation of I B, nuclear translocation of NFB subunits p50 and p65, and the upregulation of MAFbx expr ession (Figures 4-20 through 4-23). Thus, it appears that NOS inhibition can prevent inflammation-associated atrophy signaling in vitro This is in agreement with the literat ure as an isoform-specific iNOS inhibitor was shown to prevent the onset of cachexia in nude mice injected with TNF(24). Further, L-NAME also completely prevented the upregulation of iNOS mRNA with stretch (Figure 4-24). The explanation for these results can be two fold. Fi rst, we know that nitric oxide concentrations increase with high magnitudes of stretch, so, elevated NO levels may be responsible for the initial act ivation and propagation of NFB signaling leading to transcription of the iNOS gene. This would suggest that either iNOS is constitutively expressed in C2C12s (which we did show with immunostaining in Figure 4-9) and wa s functional during strain prior to transcriptional regulation by NFB, or that the initial burst of NO following stretch was not due to increased iNOS activitiy but rather to that of nNOS or eNOS. Second, the TNFconverting enzyme (TACE) is mechanosensitive and may initiate activation of TNFand subsequently, NFB after stretch, either independently or in combination with NO. The ensuing iNOS transcription and NO producti on would contribute to a positiv e feedback loop to propagate the signal as long as the cyclic strain continues. However, be cause we did not manipulate TACE or measure TNFlevels, we cannot be certain of their contribution in our model. Inducible NOS Is Not Solely Responsible for NO Production during High Strain Genetic knockout of the iNOS gene in primary cultured myotubes showed blunted NFB nuclear translocation during 18% cyclic strain, but concomitant administration of L-NAME to

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117 iNOS(-/-) myotubes further inhibited th e pathway (Figures 4-26 thr ough 4-28). This suggests that either nNOS or eNOS may also contribute to the production of nitr ic oxide involved in inflammation-associated signaling. It is logical to implicate nNOS he re, since it is located within the DGC and susceptible to activation during ch anges in muscle length. On the other hand, nonisoform specific NOS inhibition did not comp letely eliminate the stretch-induced NFB activity with stretch in C2C12 myotubes, indicating that another pathway must play a role in NFB induction during high strain. Altoge ther, it is likely that two mechanosensitive enzymes, TACE and NOS, initiate the cellular events (via NO and TNF) leading to the production of iNOS and subsequently more NO which has been deem ed necessary for cachexia and inflammationassociated atrophy (24). Limitations and Future Directions Deformation induced signaling through stretc h is unquestionably diffe rent than signaling associated with active force generation. Active force generation induces metabolic, calcium, and likely more oxidative stresses than mechanical stretch alone. The varied aspects of the cellular environment have synergistic actions. Thus, it is important to consider the entire milieu when evaluating skeletal muscle responses to stress. We are aware that our culture model does not include innervation. In fact, ne rve activity and many other potent ial extraneous variables were controlled by excluding them from study and manipulating only cell loadi ng or stretch. We are confident that our findings are due to loading differences. Future studies should examine the interaction of these load-specific effects with other changes, such as denervation and hindlimb unloading or immobillization. Conclusions In conclusion, cyclic m echanical stretch can be used as a model of activity and strain in cultured skeletal muscle myotubes. First, cyclic stretch increases myotube size through induction

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118 of Akt signaling that is dependent on NOS ac tivity. Cessation of stretch causes protein degradation, altered nNOS loca lization, and a reduction in myot ube size via downr egulation of Akt, which may contribute to NFB signaling through an alternative pathway (Figure 5-1). Secondly, high magnitude cyclic strain can induce the classical pathway of NFB signaling and upregulate iNOS and MAFbx expression in cultured skeletal muscle myotubes (Figure 5-2). This study provides evidence that altered loading conditions in vitro can be used to gain a better understanding of skeletal muscle atrophy signaling as it occurs in vivo and may provide hope for finding potential remedies for th is debilitating disease.

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119 Figure 5-1. Proposed mechanism of nNOS contribution to skeletal muscle disuse atrophy signaling. The release of low levels of n itric oxide after mode rate stretch of C2C12 myotubes activates anabolic signaling through Akt and NFB survival pathways. Cessation of moderate stretch remo ves the Akt inhibition of GSK-3 and FOXO3a and promotes protein degradation vi a the alternative pathway of NFB signaling.

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120 Figure 5-2. Proposed mechanism of NOS involvement in inflammation-associated atrophy during high strain. An 18% stretch of C2C12 myotubes activates the classical pathway of NFB signaling by mechanosensitive stimulation of NOS and TNFconverting enzyme (TACE). NFB binding to promoter sites of iNOS and E3 ligases contributes to protein degrad ation and a positive feedback loop of iNOS activation if high stretch is sustained.

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139 BIOGRAPHICAL SKETCH Quinlyn Soltow is the daughter of Kimberly Bonner and Frederick Soltow Jr. Quinlyn was born and raised in Canaan Valley, WV, wh ere she attended Tucker County High School and graduated at the top of her class in 1999. Quinl yn then moved to Atlanta, Georgia, where she received her joint BS/MS degree with high honor s in 2003 from Emory University. There she studied inorganic chemistry under the advisement of Dr. Luigi Marzilli. Her masters thesis was titled Spectroscopic studies of tris(4-sulfonatophenyl) porph yrin and the binding to human serum albumin. Quinlyn chose to continue resear ch with her pursuit of a doctoral degree at the University of Florida in exercise physiology. Fo r the next 5 years, Quinlyn studied muscle physiology under Dr. David Criswell. In 2008, Quin lyn obtained her terminal degree and plans to continue her research aspira tions by completing a postdoctoral fellow position with Dr. Dean Jones at the Emory Univer sity School of Medicine.