<%BANNER%>

Effects of Nitric Oxide on Calcium-Induced Skeletal Muscle Atrophy

Permanent Link: http://ufdc.ufl.edu/UFE0041329/00001

Material Information

Title: Effects of Nitric Oxide on Calcium-Induced Skeletal Muscle Atrophy
Physical Description: 1 online resource (81 p.)
Language: english
Creator: Zeanah, Elizabeth
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: atrophy, calpains, l6, muscle, nitric, oxide
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Applied Physiology and Kinesiology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Skeletal muscle atrophy occurs as a result of long periods of disuse, as seen with bedrest, limb immobilization, and space flight; and disease, as seen with cancer, sepsis, and metabolic dysfunction. Atrophy proceeds via different mechanistic pathways among these conditions, but in most cases, including disuse, levels of intracellular calcium increase in the muscle cells. This rise in intracellular calcium stimulates calpain activation and subsequent protein degradation, or proteolysis. Calpain cleaves the structural proteins that hold the sarcomere together, thereby releasing the contractile myofibrillar proteins for proteasome degradation; this is thought to be the rate-limiting step in skeletal muscle proteolysis because the proteasome cannot degrade intact sarcomeres. Little is known about the regulation of calpain activity, however, nitric oxide has been proposed as a possible mediator. This study used the calcium ionophore calcimycin to increase intracellular calcium and induce calpain activity in L6 myotubes. It is the first to examine the effects of exogenous nitric oxide on calpain activity in mature myotubes and to demonstrate a dose-dependent protective role for nitric oxide on calpain targets and concurrent prevention of myotube atrophy. Nitric oxide attenuated calpain cleavage of the structural protein talin and reduced visible myotube atrophy. Therefore, moderate doses of nitric oxide can prevent protein degradation and atrophy following a calcium challenge in L6 myotubes.
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 Elizabeth Zeanah.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Criswell, David S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041329:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041329/00001

Material Information

Title: Effects of Nitric Oxide on Calcium-Induced Skeletal Muscle Atrophy
Physical Description: 1 online resource (81 p.)
Language: english
Creator: Zeanah, Elizabeth
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: atrophy, calpains, l6, muscle, nitric, oxide
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Applied Physiology and Kinesiology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Skeletal muscle atrophy occurs as a result of long periods of disuse, as seen with bedrest, limb immobilization, and space flight; and disease, as seen with cancer, sepsis, and metabolic dysfunction. Atrophy proceeds via different mechanistic pathways among these conditions, but in most cases, including disuse, levels of intracellular calcium increase in the muscle cells. This rise in intracellular calcium stimulates calpain activation and subsequent protein degradation, or proteolysis. Calpain cleaves the structural proteins that hold the sarcomere together, thereby releasing the contractile myofibrillar proteins for proteasome degradation; this is thought to be the rate-limiting step in skeletal muscle proteolysis because the proteasome cannot degrade intact sarcomeres. Little is known about the regulation of calpain activity, however, nitric oxide has been proposed as a possible mediator. This study used the calcium ionophore calcimycin to increase intracellular calcium and induce calpain activity in L6 myotubes. It is the first to examine the effects of exogenous nitric oxide on calpain activity in mature myotubes and to demonstrate a dose-dependent protective role for nitric oxide on calpain targets and concurrent prevention of myotube atrophy. Nitric oxide attenuated calpain cleavage of the structural protein talin and reduced visible myotube atrophy. Therefore, moderate doses of nitric oxide can prevent protein degradation and atrophy following a calcium challenge in L6 myotubes.
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 Elizabeth Zeanah.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Criswell, David S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041329:00001


This item has the following downloads:


Full Text

PAGE 1

1 EFFECTS OF NITRIC OXIDE ON CALCIUM INDUCED SKELETAL MUSCLE ATROPHY By ELIZABETH HENDERSON ZEANAH A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE D EGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

PAGE 2

2 2009 Elizabeth Henderson Zeanah

PAGE 3

3 To my parents, whose absence has not diminished their influence

PAGE 4

4 ACKNOWLEDGMENTS First and foremost, I thank my advisor Dr. David Criswell for his support, kindness, and patience during my time as a graduate student in his laboratory. Dr. Criswells loyalty and integrity as a mentor have been as impactful on my graduate studies as has been his scientific guidance. I also thank my committee members, Drs. Scott Powers and Stephen Dodd for their support. Their dedication to and passion for skeletal muscle research, expressed through their own research, teaching, and publications, have been instrumental in developing m y interest and expertise in this area. Additionally, I thank my doctoral program advisor Dr. Randy Braith for his support of my concurrent work in Dr. Criswells laboratory and the completion of this project. Second, I would like to thank Dr. Quinlyn Solt ow, formerly of the Molecular Physiology lab, for her invaluable instruction and guidance throughout all phases of this project. Her assistance and input greatly benefitted not only this project but also the development of my skills as an independent scie ntist nearly everything I know about benchwork I owe to Dr. Soltow. But above all else I thank her for being my friend. I also would like to acknowledge all of the current and past members of the Molecular Physiology lab, particularly Dr. Vitor Lira fo r his advice regarding L6 cell culture and assistance in the lab. Finally, I thank my family and friends for their support over the past three years. I would not have had the strength and motivation to pursue this degree through some of the most difficult of adversities were it not for their love and encouragement.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF FIGURES .............................................................................................................................. 7 LIST OF ABBREVIATIONS .............................................................................................................. 9 ABSTRACT ........................................................................................................................................ 10 CHAPTER 1 INTRODUCTION ....................................................................................................................... 11 Background .................................................................................................................................. 11 Specific Aims and Hypotheses ................................................................................................... 13 Clinical Significance ................................................................................................................... 13 Strengths and Limitations ........................................................................................................... 14 2 LITERATURE REVIEW ........................................................................................................... 16 Overview of Skeletal Muscle Atrophy ...................................................................................... 16 Role of Lysosomes .............................................................................................................. 16 Role of Proteasomes ............................................................................................................ 17 Ubiquitin -dependent pr oteolysis ................................................................................. 18 Atrophyassociated E3 ligases and relationship with the IGF 1/Akt pathway via FOXO .................................................................................................................. 18 Ubiquitin independent proteol ysis .............................................................................. 19 Limitations of the proteasome system ........................................................................ 19 Role of Caspases .................................................................................................................. 19 Caspase Regulation and Calpains ....................................................................................... 20 Overview of Calpains .................................................................................................................. 20 Regulation of Calpains ................................................................................................................ 21 Intracellular Ca2+.................................................................................................................. 21 Calpastatin ............................................................................................................................ 22 Overview of NO Production in Skeletal Muscle ....................................................................... 23 Evidence for NO as a Potential Regulator of Calpains ............................................................. 23 Tissues Other than Skeletal Muscle .................................................................................... 24 Skeletal Muscle .................................................................................................................... 24 Summary ...................................................................................................................................... 25 3 MATERIALS AND METHODS ............................................................................................... 27 Experimental Designs ................................................................................................................. 27 Experiment 1 Design Aim 1, Hypothesis 1A (Figure 3 1) ............................................. 28 Experiment 2 Design Aim 1, Hypothesis 1B (Figure 3 2) ............................................. 28

PAGE 6

6 Experiment 3 Design Aim 1, Hypothesis 1B (Figure 3 3) ............................................ 29 Experiment 4 Design Aim 2, Hypothesis 2A (Figure 3 4) ............................................ 30 Experiment 5 Design Aim 2, Hypothesis 2B (Figure 3 5) ............................................ 30 General Methods ......................................................................................................................... 31 Myogenic Culture ................................................................................................................ 31 Image Analysis ..................................................................................................................... 31 Whole Cell Lysate ............................................................................................................... 32 Western Blot Analysis ......................................................................................................... 32 Statistical Analysis ...................................................................................................................... 33 4 RESULTS .................................................................................................................................... 39 Effects of Treatment with A23187 and NO on Molecular Markers ........................................ 39 Talin ...................................................................................................................................... 39 Akt ........................................................................................................................................ 40 FOXO3a ............................................................................................................................... 40 MAFbx ................................................................................................................................. 41 Effects of A23187 Treatment and NO on Myotube Atrophy ................................................... 41 Protein Degradation ............................................................................................................. 41 Myotube Image Analysis .................................................................................................... 42 5 DISCUSSION .............................................................................................................................. 65 Main Findings .............................................................................................................................. 65 A23187 Causes Degradation of Intermediate Filaments by Calpains Independent of Proteasome Activity ......................................................................................................... 65 Exogenous NO Prevents Degradation of Intermediate Filaments by Calpains Independent of Proteasome Activity............................................................................... 69 High Doses of A23187 Cause L6 Myotube Atrophy ........................................................ 72 Exogenous NO Protects L6 Myotubes from A23187Induced Atrophy .......................... 73 Limitations and Future Directions ............................................................................................. 74 Conclusions ................................................................................................................................. 75 LIST OF REFERENCES ................................................................................................................... 77 BIOGRAPHICAL SKETCH ............................................................................................................. 81

PAGE 7

7 LIST OF FIGURES Figure page 3 1 Experiment 1 design Ai m 1, Hypothesis 1A ..................................................................... 34 3 2 Experiment 2 d esign Aim 1, H ypothesis 1B ..................................................................... 35 3 3 Experiment 3 design Aim 1, Hypothesis 1B ..................................................................... 36 3 4 Experiment 4 design Aim 2, Hypothesis 2A ..................................................................... 37 3 5 Experiment 5 design Aim 2, Hypothesis 2B ...................................................................... 38 4 1 Talin cleavage in L6 myotubes after 60 minutes of treatment with A23187 ..................... 45 4 2 Talin cleavage in L6 myotubes after 60 minutes of treatment with A23187 and/or PAPA NO or L NMMA pre -conditioning ........................................................................... 46 4 3 Talin cleavage in L6 myotubes after 60 minutes of treatment with 20 M A23187 and/or PAPA NO pre -conditioning with and without calpeptin ......................................... 47 4 4 Phopho/Total Akt protein expression in L6 myotubes after 30 or 60 minutes of treatment with A23187 .......................................................................................................... 48 4 5 Phospho/Total Akt protein expression in L6 myotubes after 60 minutes of treatment with A23187 and/or PAPA NO or L NMMA pre -conditioning ......................................... 49 4 6 Phospho/Total Akt protein expression in L6 myotubes after 60 minutes of treatment with 20 M A23187 and/or PAPA NO pre -conditioning with and without calpeptin...... 50 4 7 PhosphoFOXO3a protein expression in L6 myotubes after 30 or 60 minutes of treatment with A23187 .......................................................................................................... 51 4 8 PhosphoFOXO3a protein expression in L6 myotubes after 60 minutes of treatment with A23187 and/or PAPA NO or L NMMA pre -conditioning ......................................... 52 4 9 PhosphoFOXO3a protein expression in L6 myotubes after 60 minutes of treatment with 20 M A23187 and/or PAP A NO pre -conditioning with and without calpeptin...... 53 4 10 MAFbx protein expression in L6 myotubes after 30 or 60 minutes of treatment with A23187 .................................................................................................................................... 54 4 11 MAFbx protein expression in L6 myotubes after 60 minutes of treatment with A23187 and/or PAPA -NO or L -NMMA pre -conditioning ................................................. 55 4 12 MAFbx protein expression in L6 myot ubes after 60 minutes of treatment with 20 M A23187 and/or PAPA -NO pre conditioning with and without calpeptin .......................... 5 6

PAGE 8

8 4 13 Total protein concentrations for L6 myotubes after 24 hours treatment wi th A23187 ..... 57 4 14 Total protein concentrations for L6 myotubes after 24 hours treatment with A23187 and/or DETA NO ................................................................................................................... 58 4 15 Repre sentative images of L6 myotubes after 24 and 48 hours treatment with A23187 .... 59 4 16 Image analysis of L6 myotubes after 24 and 48 hours treatment with A23187................. 60 4 17 Representative images of L6 myotubes after 24 hours treatment with A23187 and/or DETA NO ............................................................................................................................... 61 4 18 Image analysis of L6 myotube diameter after 24 hours trea tment with A23187 and/or DETA NO ............................................................................................................................... 62 4 19 Image analysis of L6 myotube length after 24 hours treatment with A23187 and/or DETA NO ............................................................................................................................... 63 4 20 Image analysis of L6 myotube area after 24 hours treatment with A23187 and/or DETA NO ............................................................................................................................... 64

PAGE 9

9 LIST OF ABBREVIATION S A23187 calcimycin, calcium ionophore A23187 Akt Akt/protein kinase B ATP adenosine triphosphate ATPase adenosine triphosphatase Ca2+ calcium ion DETA NO diethylenetriamine NONOate DMSO dimethyl sulfoxide EDTA ethylenediaminetetraacetic acid eNOS endothelial nitric oxide synthase FOXO forkhead box O IGF 1 insulin like growth factor 1 iNOS induc ible nitric oxide synthase L -NMMA L -NG-monomethyl arginine citrate MAFbx muscle atrophy F -box MuRF1 muscle ring finger 1 nNOS neuronal nitric oxide synthase NO nitric oxide NOS nitric oxide synthase PAPA NO propylamine propylamine NONOate PBS phosphate -buf fered saline PI3K phosphoinositide 3 kinase PVDF polyvinylidene difluoride TBS tris buffered saline

PAGE 10

10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF NITRIC OXIDE ON CALCIUM INDUCED SKELETAL MUSCLE ATROPHY By Elizabeth Henderson Zeanah December 2009 Chair: David S. Criswell Major: Applied Physiology and Kinesiology Skeletal muscle atrophy occurs as a result of long pe riods of disuse, as seen with bedrest, limb immobilization, and space flight; and disease, as seen with cancer, sepsis, and metabolic dysfunction. Atrophy proceeds via different mechanistic pathways among these conditions, but in most cases, including dis use, levels of intracellular calcium increase in the muscle cells. This rise in intracellular calcium stimulates calpain activation and subsequent protein degradation, or proteolysis. Calpain cleaves the structural proteins that hold the sarcomere togeth er, thereby releasing the contractile myofibrillar proteins for proteasome degradation; this is thought to be the rate limiting step in skeletal muscle proteolysis because the proteasome cannot degrade intact sarcomeres. Little is known about the regulati on of calpain activity, however, nitric oxide has been proposed as a possible mediator. This study used the calcium ionophore calcimycin to increase intracellular calcium and induce calpain activity i n L6 myotubes. It is the first to examine the effects of exogenous nitric oxide on calpain activity in mature myotubes and to demonstrate a dose -dependent protective role for nitric oxide on calpain targets and concurrent prevention of myotube atrophy. Nitric oxide attenuated calpain cleavage of the structur al protein talin and reduced visible myotube atrophy Therefore, moderate doses of nitric oxide can prevent protein degradation and atrophy following a calcium challenge in L6 myotubes.

PAGE 11

11 CHAPTER 1 INTRODUCTION Background Skeletal muscle atrophy can result from a variety of conditions, such as prolonged bed rest, limb immobilization, denervation, cancer, sepsis, aging, malnutrition, metabolic dysfunction, and space flight. Skeletal muscle atrophy is a clinically -significant problem not only because it is a common complication and/or outcome of these conditions but also because it can contribute to the onset or worsening of others. Loss of muscle mass is a primary cause of decreased mobility and balance, which can lead to inactivity, injury, and inability to perform daily tasks, as well as contribute to metabolic syndrome (obesity, hyperlipidemia, hypertension, and insulin resistance) and increased risk for type II diabetes and cardiovascular disease. While there are several different molecular pathways th rough which skeletal muscle atrophy can occur depending upon the atrophy stimulus, elevated intracellular Ca2+ occurs in most atrophic conditions. The role of Ca2+ is especially critical to understanding disuse atrophy because Ca2+ levels greatly increase with disuse (10). The mechanism for this rise in Ca2+ is unknown, but increasing oxidative stress may play a major role ( 27). Elevated intracellular Ca2+ is a powerful atrophy signal because Ca2+ stimulates calpain activity. Calpains are Ca2+dependent cysteine proteases that cleave the intermediate filaments of the sarcomere to release actin and myosin for degradation by the proteasome (6 ). Calpains cannot degrade contractile proteins, but, because myofibrillar release must precede proteasome proteoly sis, calpain activity is often cited as the rate limiting step in proteolysis ( 27). In fact, evidence from rat diaphragm muscle shows that calpains may be necessary for protein degradation by the proteasome ( 31). Interestingly, the same study has present ed evidence that calpain activity inhibits protein synthesis via the IGF 1/Akt pathway, suggesting a dual role for calpains in muscle atrophy ( 31).

PAGE 12

12 Little is known about the regulation of calpain activity. However, a small number of research studies have suggested NO as a possible mediator. NO has been shown to inhibit calpain activity in human neutrophils ( 22) and rat cardiac myocytes ( 5 ). Evidence in skeletal muscle is limited to date. In C2C12 myotubes, mechanically induced endogenous NO release has been associated with increased intact intermediate filaments known to be cleaved by calpains (42). And in C2C12 myoblasts, induction of calpain activity with A23187 produced intermediate filament cleavage, and this activity was attenuated by exogenous NO (19). This is the strongest evidence to date of a protective role for NO in calpain proteolysis in skeletal muscle. However, this relationship has not yet been established in mature myotubes and has not been connected with other atrophy markers. Thus, s everal questions remain regarding the role of NO in regulating calpain activity in skeletal muscle. First, it is unknown if exogenous NO will directly inhibit calpain action in mature myotubes. Second, it is unknown whether or not the effects of NO are d ose -dependent. Third, even if exogenous NO does decrease the cleavage of intermediate filaments by inhibiting calpains, it has not been clearly established that this prevents muscle atrophy (loss of myotube size and/or total protein). None of the aforeme ntioned studies has presented clear evidence of atrophy in connection with calpain induction or prevention of atrophy with an increase in NO. While this relationship between calpain proteolysis and muscle atrophy may seem obvious, it is possible that at least some of the calpain activity is devoted to cellular remodeling. Thus, evidence of myotube atrophy and/or activity of other atrophyassociated proteins, such as the proteasome E3 ligases MAFbx and MuRF1, are needed in conjunction with evidence of calp ain proteolysis and NO effects

PAGE 13

13 Specific Aims and Hypotheses Specific aim 1: To test that elevated intracellular Ca2+ levels induced by treatment with A23187 is a dose -dependent model of skeletal muscle atrophy stimulating calpain proteolysis of intermedi ate filaments and increasing molecular markers of proteasome activity and to determine whether or not exogenous NO can attenuate these effects. Hypothesis 1A: Elevation of intracellular Ca2+ by treatment with A23187 causes increased proteolysis of intermed iate filaments (e.g. talin) by calpain and increased proteasome activity (e.g. FOXO3a, MAFbx). Hypothesis 1B: Exogenous NO administration attenuates calpain proteoloysis and proteasome activity after treatment with A23187. Specific aim 2: To test if elevat ed intracellular Ca2+ by treatment with A23187 induces myotube atrophy (loss of size and/or total protein) and whether or not exogenous NO administration results in decreased atrophy. Hypothesis 2A: Elevation of intracellular Ca2+ by treatment with A23187 causes myotube atrophy. Hypothesis 2B: Exogenous NO attenuates myotube atrophy after treatment with A23187. Clinical Significance Skeletal muscle accounts for approximately 40% of human body mass and is required for basic functions such as locomotion, resp iration, metabolism, and thermoregulation. Skeletal muscle responds to the presence or absence of stimuli with changes in protein synthesis, protein degradation, or both. When the rate of protein degradation surpasses the rate of protein synthesis, skelet al muscle atrophy occurs. Atrophy is described as a loss in muscle fiber size, protein content, and strength. An increase in protein degradation and subsequent muscle atrophy can occur due to periods of inactivity, lack of nutrition, aging, or disease a nd can

PAGE 14

14 contribute to numerous disease states. A loss of skeletal muscle leads to fatigability, decreased mobility and ability to execute daily activities, and insulin resistance, all of which are risk factors for type II diabetes and cardiovascular diseas e. Skeletal muscle loss is also a complication in cancer and cancer therapies. Much work has been done in the field of skeletal muscle atrophy. Various models, including limb immobilization, limb unloading, denervation, and endotoxin administration, have been used to research the causes of muscle atrophy. These models are all performed in vivo ; they can provide little insight on the intrinsic factors leading to muscle degradation and are heavily influenced by many neural and humoral factors. Therefore, it is necessary to study skeletal muscle atrophy in vitro to investigate the signaling pathways leading to loss of muscle mass and what might attenuate atrophy signaling. Elucidating these signals and what mediates them may lead to therapeutic strategies, su ch as pharmaceutical interventions, to attenuate or eliminate skeletal muscle atrophy as a complication and cause of disease, which could dramatically improve both longevity and quality of life. Strengths and Limitations The primary strength of this in vitro study is the high level of experimental control exercised over the muscle cells. Unlike in vivo studies, this study was not subject to influence by the many humoral and neural factors that would be confounding in a whole animal. Although in vitro stud ies require confirmation in vivo this study provides one piece of the cellular signaling puzzle that would be impossible to ascertain without in vitro experimentation that eliminates other physiological factors. Furthermore, this is the first study of ca lpain proteolysis and NO mediation that examines not only molecular signals associate d with proteolysis but also the degree of atrophy in the myotubes.

PAGE 15

15 This study used A23187 to induce skeletal muscle atrophy because elevated intracellular Ca2+ is a common element in most, if not all, models of skeletal muscle atrophy and is particularly cha racteristic of disuse atrophy (10). While this stimulus is a useful simulation of skeletal muscle atrophy in general, this model cannot be equated with a specific atrop hy stimulus. And although myotube atrophy was induced and a dose -response experiment revealed a trend of increasing atrophy signals with A23187, it is not certain exactly which atrophy pathways were activated. This study examined talin cleavage as an ind irect measure of calpain activity, MAFbx expression as an indicator of proteasome activity, FOXO3a as a stimulator of MAFbx transcription and Akt phosphorylation as an inhibitor of FOXO3a activity However, elucidating all of the pathways involved was be yond the scope of this study Additionally, it is possible that some of the atrophy signals were missed due to the time points selected for study. Cellular protein was harvested after 60 minutes of incubation with A23187 based upon previous evidence that proteasome activity in L6 myotubes increases most rapidly during the first 40 minutes of incubation with A23187 and then continues to increase significantly for 120 minutes ( 21). It was believed that 60 minutes would be sufficient time to see proteasome a ctivity but still brief enough not to miss evidence of the calpain cleavage that should precede it. Incubating for a longer period would have risked losing the cleaved talin protein to proteasome degradation, and since this study was primarily concerned w ith calpain activity in this study, the time was limited to 60 minutes. However, experiments done to show myotube atrophy were conducted for both 24 and 48 hours, demonstrating that the effects of A23187 and NO were not transient.

PAGE 16

16 CHAPTER 2 LITERATURE REV IEW Skeletal muscle atrophy is a clinically significant problem as it arises from a variety of factors, such as prolonged bed rest, limb immobilization, space flight, denervation, cancer, sepsis, and ageing. Loss of muscle mass has been shown to contribute to inactivity, obesity, insulin resistance, hypertension, and hyperlipidemia (altogether termed metabolic syndrome). Minimization of muscle atrophy and its side effects will prevent the progression to more serious pathological disorders such as metabolic disease and loss of functional independence and overall quality of life. Overview of Skeletal Muscle Atrophy During atrophy the rate of protein degradation exceeds the rate of protein synthesis. Both degradation and synthesis are highly complex, intricate ly regulated cellular processes that can occur via many interdependent pathways. However, it is generally accepted that in muscle atrophy, the increase in proteolysis has a greater impact than protein synthesis ( 27). There are four major proteolytic syst ems in skeletal muscle: lysosomes, proteasomes, caspases, and calpains. Role of Lysosomes Lysosomes are highlyacidic, membrane -bound vesicles containing proteases, lipases, phosphatases, and other catabolic molecules. The dominant lysosomal proteases are cathepsins L, B, D, and H (2). Although these cathepsins are found in all tissues, the relative degree of their expression is highly correlated with the tissues inherent rate of protein turnover (2). Because skeletal muscle has a relatively low rate of protein turnover, cathepsin concentrations are relatively low in skeletal muscle (2). This may explain, at least in part, why cathepsins seem to have a small role in skeletal muscle atrophy. Inhibition of lysosome and/or cathepsin activity

PAGE 17

17 does not sign ificantly attenuate either proteolysis or atrophy (9 34, 37). Similar evidence suggests that the cathepsins do not degrade myofibrils but rather are responsible for degrading membrane proteins ( 18). While the import of membrane proteolysis cannot be dis missed, this action is not sufficient to produce atrophy. However, s everal experiments have shown that cathepsin mRNA expression and/or cathepsin activity increase(s) during dis use atrophy (reviewed in ref. 18 ). This may be evidence of cell maintenance and healthy protein turnover. Lysosomes are important mediators of autophagy, which is a mechanism for removal of old or damaged cellular proteins or organelles. In this process, proteins or cellular components targeted for removal are imported into, or e ngulfed by lysosomes. Although autophagy is important in striated muscle, recent evidence shows that increased autophagy is associated with improved cellular function and protect ion against age associated atrophy ( 3, 1 8 ). Thus, we can conclude that the lys osomal proteases are significant yet minor pla yers in skeletal muscle atrophy and may even protect against age associated atrophy. Since they do not appear to degrade the myofibrils, a more dominant system must be involved in atrophic proteolysis. Role of Proteasomes Proteasomes are generally accepted to be responsible for the bulk of skeletal muscle proteolysis because they degrade the contractile myofibrillar prot eins (i.e. actin and myosin) (18, 27). Protein degradation by the proteasomes can occur in two ways. First, the 26S proteasome can degrade ubiquitinated proteins in an energy-dependent process. The 20S proteasome, however, can degrade proteins in an energy and ubiquitin independent process.

PAGE 18

18 Ubiquitin dependent proteolysis Ubiquitin -dependent pr oteolysis requires ATP and the synergistic action of the two components of the 26S proteasome: the 20S proteolytic core and the 19S regulatory subunits. The 26S proteasome is made up of a 20S proteasome and 19S regulatory subunits with the high ATPase act ivity necessary for energy -dependent proteasome action (26, 27, 35). These 19S complexes are responsible for recognizing and binding ubiquitinated proteins, removing the ubiquitin chain through ATP hydrolysis, and transferring the protein into the 20S proteasome core for passive degradation (27, 35). Ubiquitin is a protein that covalently bonds to targeted proteins through an ubiquitin protein ligating system ( 35). This system consists of an ubiquitinactivating enzyme (E1), several ubiquitin-conjugating enzymes (E2), and substrate -specific ubiquitin ligating enzymes (E3) (17). All three enzymes are required for ubiquitin -proteasome action. At least one ubiquitin -conjugating enzyme, E214k, is significant in regulating skeletal muscle proteolysis ( 20). H owever, because the E3 enzymes are responsible for selective protein recognition and are substrate -specific, they appear to be especially important in the regulation of tissue -specific atrophy ( 13, 35). Atrophy associated E3 ligases and relationship with t he IGF -1/Akt pathway via FOXO While many E3 ubiquitin ligating enzymes have been identified, the gene expressions of two specific E3s have been identified as being upregulated during skeletal muscle atrophy: MuRF1 and MAFbx, also known as atrogin1 ( 12). These genes are particularly significant because they are up regulated in nearly all models of atrophy, including unweighting, immobili zation, denervation, cachexia (4 ), starvation ( 15), and sepsis ( 41). Both MAFbx and MURF 1 have been implicated in at least 13 atrophy model s, including models of disuse (4, 13), but their upregulation can be antagonized by simultaneous treatment with IGF 1 (28 ) acting

PAGE 19

19 through the PI3K/Akt pathway (29, 33). This suggests a novel role for Akt inhibition of atrophy signaling. The mechanism by which Ak t inhibits MAFbx and MuRF1 upregulation involves the FOXO family of transcription factors (29, 33). 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 induce atrophy (30 ). Ubiquitin -independent proteolysis Recent evidence supports the existence of an ubiquitinindependent proteolytic process as well. The 20S proteasome can recognize and degrade oxidized proteins without the 19S subunits and ubiquitin ligase chain ( 16). Furthermore, this pathway obviates the need for ATPases because 20S proteosome degr adation is a passive pro cess (16 27). These findings suggest that oxidative stress is a mediator of skeletal muscle atrophy dri ven by the proteasome system (27). Limitations of the proteasome system Because of their collective ability to degrade the contractile proteins, the pr oteasomes are responsible for the bulk of skeletal muscle proteolysis. However, proteasomes cannot degrade intact sarcomeres ( 14). Since the majority of muscle proteins are contained within these actomyosin complexes, there must be additional pathways by which the myofibrils are released prior to proteasome degradation ( 36). This myofibrillar release is performed by two interrelated but distinct Ca2+-dependent proteolytic systems: caspases and calpains. Role of Caspases Caspases are Ca2+-dependent cys teine proteases capable of cleaving the intermediate filaments of the sarcomeres and releasing myofibrils for degradation by the proteasomes (8, 14, 36). Furthermore, inhibition of caspase 3 prevents actin accumulation in the cytosol, indicating that casp ase 3 is a necessary first step fo r skeletal muscle proteolysis (8 ).

PAGE 20

20 However, caspases are best known for their role in triggering apoptosis (6). Apoptosis results in a loss of myonuclei (and likely myofiber size as a result) and occurs through at least t hree known pathways: sarcoplasmic reticulum, receptor -mediated, and mi tochondrial (reviewed in ref. 27 ). Caspase Regulation and Calpains In the sarcoplasmic reticulum pathway, another proteolytic system, the calpains, partially mediates caspase activity. When the sarcoplasmic reticulum is injured, the regulation of Ca2+ release and sequestration is impaired, and Ca2+ accumulates in the cytosol (27). The high level of Ca2+ activates both caspase 7 and calpain, either of which can begin a caspase cascade leading to nuclear dam age and subsequent apoptosis (27). So, calpain activation appears to promote caspase apoptotic activity. Also, caspase expression appears to enhance calpain activity by serving as a substrate for calpastatin, the only endogenous inhibitor of the calpains (reviewed in a subsequent section). Thus, when caspases are highly expressed, calpastatin activity will decrease as it binds to caspase substrates, and calpain activity will then increase in the absence of its inhibitor. Calpains, the fourth proteolytic system in skeletal muscle, are fully dicussed in the following section. Overview of Calpains Calpains, like caspases, are Ca2+dependent cysteine proteases that cleave the intermediate filaments of the sarcomere to release actin and myos in for proteasome degradation (6 ). They are ubiquitously expressed in all mammalian cells, but some isoforms are tissue -specific (6 ). Calpains cannot degrade contractile proteins but, because this myofibrillar release must precede proteasome proteoly sis, calpain activity is often cited as the rate -limiting step in proteolysis ( 27). (Recent literature has also proffered non proteolytic roles of the calpains as well, including

PAGE 21

21 involvement in proliferation, differentiation, migration, and gene expression, but these are beyond the scope of this review.) Calpains appear to be necessary for proteasome protein degradation. Recent ex vivo experiments in the rat diaphragm showed that calpain activation increased total proteolysis by 65% and proteasome -depende nt prote olysis by an impressive 144% (31 ). In the same experiments, inhibiting the proteasome during calpain activation prevented this increase in proteolysis ( 31). Taken together, this data supports the theory of sequential proteolysis just described an d illustrates the necessity of both calpains and proteasomes in skeletal muscle atrophy. Interestingly, calpain activity concurrently inhibited protein synthesis via the IGF 1/Akt pathway, suggesting a dual role for calpains in muscle atrophy ( 31). At lea st 15 distinct calpain isoforms have been identified (6 ). Skeletal muscle expresses three dominant isoforms: calpain 1 ( -calpain), calpain 2 (m -cal pain), and calpain 3 (p94) (1, 6, 38). Calpains 1 and 2 are implicated in skeletal muscle atrophy (1, 6, 2 7, 32) and exist as heterodimers (6 ). Regulation of Calpains Although the regulation of calpains has yet to be fully elucidated, intracellular Ca2+ and calpastatin have been shown to exert great influence over calpain activity. Intracellular Ca2+ Intrace llular Ca2+ is a powerful stimulator of calpain activity. As intracellular Ca2+ concentrations rise, calpain moves to the plasma membrane for activation. There, Ca2+ and membrane phospholipids activate the calpain heterodimer, which than dissociates into a ctive calpain 1 and calpain 2 (6 ). The role of Ca2+ is critical to understanding disuse atrophy because Ca2+ levels increase with disuse (10). The mechanism for this rise in Ca2+ is unknown, but increasing oxidative stress

PAGE 22

22 may play a major role (27). R eactive oxygen species can damage cellular membranes and the sarcoplasmic reticulum, increasing permeability and Ca2+ leakage (10, 27). Ca2+ may also leak from the mitoch ondria, worsening the problem (10 ). So, the high levels of intracellular Ca2+ seen d uring disuse atrophy support the idea that calpains are crucial contributors in these atrophy models. However, the levels of Ca2+ needed to achieve half -maximal proteolysis by calpain 1 and calpain 2 are much higher than physiological Ca2+ levels (38). Du ring normal function, the sarcoplasmic reticulum maintains Ca2+ concentrations at approximately 107 M (11 ). And even during atrophy, Ca2+ concentrations are unlikely to reach the 0.2 1 mM needed to activate calpain 2 or even the 2 75 M ne eded for calpai n 1 activation (6, 38). But Ca2+ somehow still activates calpains as evidenced by the strong body of literature supporting calpain proteolysis. Several hypotheses have been offered to explain physiological Ca2+ activation of calpain, including increased Ca2+ affinity in vivo due to interaction wi th calpain activator proteins (6 ). The exact mechanism for calpain activation in relatively low Ca2+ concentrations, however, remains unknown. Calpastatin Calpastatin is the only known endogenous inhibitor of cal pain (36 ). Calpastatin binds to calpain and thereby prevents it from cleavin g the intermediate filaments (26 ). So, calpain activity appears to be mediated by a balance between intracellular Ca2+ concentrations and calpastatin expression. Calpastatin has been used to study calpain activity in many atrophy models. Transgenic mice that overexpress human calpastatin significantly increased soleus and extensor digitorum longus muscle mass in ex vivo experiments (24 ). During hindlimb unloading, overexpression of

PAGE 23

23 calpastatin reduced muscle atrophy by a significant 30% (36). This evidence points to not only the power of calpastatin as an inhibitor but also the requisite role of calpain activity in muscle atrophy. Overview of NO Production in Skeletal Muscle NO is a gaseous free radical with many biological effects. Due to its high chemical reactivity, NO can be a powerful signaling molecule and antioxidant or can be harmful through the nitrosylation of many proteins. NO is produced physiologically by the trans formation of L arginine by three NOS isoforms; all three isoforms catalyze the formation of NO from L arginine, oxygen, and NADPH. In skeletal muscle, two isoforms are constituitive: eNOS and nNOS (23 ). The third isoform, iNOS, is induced by cytokines and is only transient ly active in skeletal muscle (23, 39 ). eNOS and nNOS are activated to produce NO during both mec hanical loading and activity (32, 39, 42). To acquire the active state, eNOS and nNOS also require calmodulin (CaM) and Ca2+, indicating tha t NO synthesis is triggered by an elevation of intracellular Ca2+ a nd that activity induced NO production, in particular, is stimulated by Ca2+. It is important to note that although Ca2+ also stimulates calpain activity, the intracellular Ca2+ accumulati on during normal activity and/or exercise is transient and therefore insufficient to promote Ca2+dependent proteolysis (reviewed in ref. 11 ). Therefore, it is still possible that muscle activity/ Ca2+dependent nitric oxide release could attenuate calpai n activity. Evidence for NO as a Potential Regulator of Calpains A limited number of studies have proposed NO as a potential regulator of calpain activity. It is difficult to draw conclusions based on the results of these studies because they vary with r espect to the source of NO, the in vitro techniques used, the species used (human, rat, mouse), and the tissue types studied.

PAGE 24

24 Tissues Other than Skeletal Muscle A decade ago Michetti et al. were the first to establish a direct connection between calpain and nitric oxide with work in human neutrophils, demonstrating that NO donor sodium nitro -prusside blocked nearly all calpain 2 activity but ha d little effect on calpain 1 (22 ). Although this work was not done in skeletal muscle, it is possible that the s imilar effects could be seen across tissue types. More recently other evidence of nitric oxide inhibition of calpain activity has been shown in cardiac myocytes. Chohan et al. discovered that exogenous L arginine administration restored SR function in intact, isolated ischemia reperfused rat hearts by preventing calpain activation (5 ). Although claiming calpain inhibition as the cause of the SR restoration is tenuous due to measurement from muscle homogenate, this study provides at least a promising indication of nitric oxides ability to inhibit calpains. Skeletal Muscle Research of NO inhibition of calpains in skeletal muscle is so far very limited. In 2004, Zhang et al. showed that stretch induced increases in nNOS expression and endogenous NO relea se were associated with increased intact talin and vinculin, two intermediate filaments known to be cleaved by calpains, and decreas ed cleaved/total talin ratio (42 ). This study suggested that NO both up-regulated talin expression in response to mechanical loading and down regulated cleavage of talin by inhibiting calpain activity ( 42). However, it is important to note that calpain inhibitors were not used in the protein expression analysis (although calpain inhibition appeared to have an impact on elasti c modulus), so it is possible that NO -induced upregulation of intermediate filament expression, and not inhibition of degradation by calpains, was responsible for the changes.

PAGE 25

25 Also recently, Koh and Tidball administered the Ca2+ ionophore A23187 to C2C12 myoblasts to induce calpain activity and showed that the NO -donor SNP prevented proteolysis of talin ( 19). The inhibition of calpain action by NO approximated the inhibition caused by a known calpain inhibitor ( 19). This is the strongest evidence to date of a protective role for NO in calpain proteolysis in skeletal muscle. However, this relationship has not yet been established in mature myotubes and has not been connected with other atrophy markers. Several questions then remain regarding the role of NO in regulating calpain activity in skeletal muscle. First, it is unknown if exogenous NO will directly inhibit calpain action in mature myotubes. Second, it is unknown whether or not this protective effect is dose -dependent. Third, even if exogenous NO does decrease the cleavage of intermediate filaments by inhibiting calpains, it has not been clearly established that this prevents muscle atrophy (loss of myotube size and/or total protein). None of the aforementioned studies has presented clear evidence of atrophy in connection with calpain induction or prevention of atrophy with an increase in NO. While this relationship between calpain proteolysis and muscle atrophy may seem obvious, it is possible that at least some of the calpain activity is devoted to cellular remodeling. Thus, evidence of visible myotube atrophy and/or activity of downstream atrophy-associated proteins, such as the proteasome E3 ligases MAFbx and MURF1 are needed. Summary Skeletal muscle atrophy occurs as a result of many conditions and involves many molecular signaling pathways. Elevated intracellular Ca2+ is present in most of these atrophy states and is definitely implicated in disuse atrophy. Ca2+ is a potent stimulator of calpains, which exert considerable control over skeletal muscle atrophy by cleaving the intermediate filaments in the sarcomere to release the contractile proteins for degradation by the proteasome. Little is known about the regulation of calpains, but NO has been suggested as a calpain

PAGE 26

26 inhibitor. If this is true in skeletal muscle, and NO does in fact inhibit calpain cleavage of intermediate filaments and subsequent proteasome action, then it could play a significant role in the prevention of muscle atrophy when Ca2+ is elevated.

PAGE 27

27 CHAPTER 3 MATERIALS AND M ETHODS Experimental Designs An immortal cell line of rat skeletal muscle cells called L6 (ATCC, Manassas, VA) was used for all experiments. The L6 myogenic cell line was isolated originally by Yaffe from primary cultures of rat thigh muscle. These cells fuse in culture to differentiate into multinucleated myotubes and are a widely used model to study differentiated skeletal muscle cells. The L6 myogenic cell line was chosen in lieu of the C2C12 myogenic cell line produced in mouse muscle because, unlike C2C12 myotubes, L6 myotubes will not contract when exposed to a Ca2+ challenge. Three experiments were performed in order to establish elevated intracellular Ca2+ levels induced by treatment with A23187 (Sigma, St. Louis, MO) as a dose -dependent model of s keletal muscle atrophy that stimulates calpain proteolysis of intermediate filaments and increases molecular markers of proteasome activity and to determine whether or not exogenous NO can attenuate these effects. Initial experiments exposed these cells t o 30 60 minutes of A23187, while cells in subsequent experiments were pre -conditioned for 60 minutes (two hours total incubation) with PAPA -NO (Cayman Chemical, Ann Arbor, MI), an NO donor with a half life of 15 minutes before treatment with A23187. Some cells were pre -conditioned with L NMMA (Cayman Chemical), a blocker of endogenous NO release. Calpeptin (Calbiochem/EMD Chemicals, Gibbstown, NJ), a known inhibitor of calpain I and II, was administered to some groups in order to compare the effects of NO with the potent but specific effects of the inhibitor. In all of these experiements, whole cell lysate was harvested from all groups and used for Western blot analysis.

PAGE 28

28 Two additional experiments were performed in order to test whether or not the model in duced visible myotube atrophy, not simply cellular re -modeling, and also to test the effects of NO on potential myotube atrophy. In these experiments cells were exposed to different doses of A23187 for a longer time period (2428 hours) in order to allow atrophy to occur. Some cells were also treated with DETA NO (Cayman Chemical), an NO donor with a half life of 20 hours. V isual images were taken for image analysis to determine changes in myotube size. Cells treated for 24 hours were also harvested and measured for total protein. Experiment 1 Design Aim 1, Hypothesis 1A (Figure 3-1) Experiment 1 was used to test elevated intracellular Ca2+ induced by treatment with A23187 as a dose -dependent model of skeletal muscle atrophy stimulating calpain proteo lysis of intermediate filaments and increasing proteasome activity. It was also used to determine the dose(s) of A23187 that would induce atrophy signals and that would be used in subsequent experiments. Previous studies administered doses ranging from 1 M to 100 M (19, 21). L6 myoblasts were cultured and differentiated into myotubes for four days before undergoing either 30or 60-minute treatments with A23187. Groups 1 4 were treated for 30 minutes with the following doses of A23187 dissolved in DMS O: 0 M (DMSO vehicle only), 1 M, 10 M, 20 M. Groups 5 8 were incubated for 60 minutes with the same doses of A23187. For all groups n = 3. After the treatment period, cells were immediately lysed and harvested, and the whole cell lysate was prepa red for Western blotting. Western blots were performed to measure cleaved and total talin, MAFbx, phospho-FOXO3a, and phophoand total Akt. Experiment 2 Design Aim 1, Hypothesis 1B (Figure 3-2) Experiment 2 was used to determine the effect of exogenou s administration of NO on calpain proteoloysis and proteasome activity after 60 minutes of treatment with A23187. L6

PAGE 29

29 myoblasts were cultured and differentiated into myotubes for four days before undergoing 60 minute treatments with A32187. Some groups we re also pre -conditioned for 60 minutes prior to the treatment period with either PAPA NO or L NMMA dissolved in NaOH. For pre conditioned groups, media was not changed between pre -conditioning and treatment, resulting in two hours of total incubation time Groups 1 3 received no A23187. Group 1 was incubated with only the DMSO vehicle, while groups 2 and 3 were pre -conditioned with PAPA -NO as follows: 1 M (group 2), 10 M PAPA NO (group 3). Groups 4 7 were treated with 10 M A23187. Group 4 received no pre -conditioning, while groups 5, 6, and 7 were pre -conditioned as follows: 1 M PAPA NO (group 5), 10 M PAPA NO (group 6), 5 mM L -NMMA (group 7). Groups 8 11 were treated with 20 M A23187. Group 8 received no pre -conditioning, while groups 9, 10, a nd 11 were pre -conditioned as follows: 1 M PAPA NO (group 9), 10 M PAPA NO (group 10), 5 mM L NMMA (group 11). For all groups n = 6. After the treatment period, cells were immediately lysed and harvested, and the whole cell lysate was prepared for West ern blotting. Total protein was measured, and Western blots were performed to probe for cleaved and total talin, MAFbx, phospho-FOXO3a, and phophoand total Akt. Experiment 3 Design Aim 1, Hypothesis 1B (Figure 3-3 ) Experiment 3 was used to compare the effects of NO on calpain proteoloysis and proteasome activity with those of calpeptin, a known calpain inhibitor, after 60 minutes of treatment with A23187. L6 myoblasts were cultured and differentiated into myotubes for four days before undergoing 60-minute treatments with A32187. Some groups were also pre conditioned for 60 minutes prior to the treatment period with either PAPA NO dissolved in NaOH and/or calpeptin dissolved in DMSO as described above. Group 1 received no treatments (DMSO vehicle o nly). Groups 2 7 were treated with 20 M A23187. Group 2 received no

PAGE 30

30 pre conditioning, while groups 3 7 were pre -conditioned as follows: 1 M NO (group 3), 10 M NO (group 4), 100 M calpeptin (group 5), 1 M NO + 100 M calpeptin (group 6), 10 M NO + 100 M calpeptin (group 7). For all groups n=6. After the treatment period, cells were immediately lysed and harvested, and the whole cell lysate was prepared for Western blotting. Western blots were performed to probe for cleaved and total talin, MAF bx, phospho-FOXO3a, and phopho and total Akt. Experiment 4 Design Aim 2, Hypothesis 2A (Figure 3 -4) Experiment 4 was used to test if elevated intracellular Ca2+ by treatment with A23187 induces visible myotube atrophy (loss of size and/or total protei n ) after 24 48 hours. L6 myoblasts were cultured and differentiated into myotubes for four days before being treated for either 24 or 48 hours with A23187. Groups 1 6 were treated for 24 hours with the following doses: 0 M (DMSO only), 0.4 M, 1 M, 5 M, 10 M, 20 M. Groups 7 12 were treated for 48 hours with the same doses. For all groups n = 3. For cells undergoing the 24 hour treatment, visual images were captured with a light microscope and analyzed immediately following treatment These cells were then harvested and measured for total protein. Cells undergoing the 48hour treatment were washed with PBS immediately following treatment and fixed with paraformaldehyde solution for visual analysis with a light microscope. Experiment 5 Design Aim 2, Hypothesis 2B (Figure 3-5) Experiment 5 was used to test the effects of NO myotube atrophy following 2448 hours of treatment with A23187. L6 myoblasts were cultured and differentiated into myotubes for four days before being treated for either 24 or 48 hours with A23187. Some groups were also treated with DETA -NO dissolved in NaOH. All of the following groups were tested at both 24 hours and 48 hours. Groups 1 3 received no A23187. Group 1 received no treatments (DMSO vehicle

PAGE 31

31 only), while groups 2 and 3 were treated with either 1 M DETA NO (group 2) or 10 M DETA NO (group 3). Groups 4 6 were treated with 10 M A23187. Group 4 received no DETA NO, while groups 5 and 6 were pre -conditioned with either 1 M DETA NO (group 5) or 10 M DETA NO (gr oup 6). Groups 79 were treated with 20 M A23187. Group 7 received no DETA NO, while groups 8 and 9 were pre -conditioned with either 1 M DETA NO (group 8) or 10 M DETA NO (group 9). For all groups n = 3. For cells undergoing the 24-hour treatment, v isual images were captured with a light microscope and analyzed immediately following treatment. These cells were then harvested and measured for total protein. Cells undergoing the 48 hour treatment were washed with PBS immediately following treatment a nd fixed with paraformaldehyde solution for visual analysis with a light microscope. General Methods Myogenic Culture Myoblasts derived from L6 cells (ATCC, Manassas, VA) were cultured on 100 mm dishes in Dulbecoos Modified Eagles Medium (DMEM) (Mediatec h, Herndon, VA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 0.1% funzigone at 37 the presence of 5% CO2 until 60 70% confluence was reached as visualized by light microscopy. The cultures were then trypsinized and r e plated at equal density to 6 -well plates for differentiation and treatment. Onc e the plate cultures reached 70 80% confluency, myotube differentiation was initiated by switching to DMEM supplemented with 2% horse serum, 1% penicillin/streptomycin, and 0. 1% fungizone. Image Analysis Three to six digital images per culture were captured using a Zeiss microscope. The images were analyzed for myotube length, diameter and area using ImageJ imaging software (NIH).

PAGE 32

32 Whole Cell Lysate Cells were harvested in ice -c old non -denaturing lysis (NDL) buffer containing 30 mM Tris HCL (pH 7.5), 0.7% Triton -X, 150 mM NaCl, 3.5 mM EDTA, 10 mg/ml NaN3 Na3VO4, 0.05% vol/vol protease inhibitors and 0.5% vol/vol phosphatase inhibitors (Sigma, St. Louis, MO). Lysates were th en centrifuged at 4C for 10 minutes at 1000 x g. Western Blot Analysis Protein concentrations were measured using the DC Protein Assay Kit (Bio -Rad Laboratories, Richmond, CA). Aliquots of whole cell lysate were run on SDS PAGE gels, and proteins were tra nsferred to either PVDF membranes (for phosphoFOXO3a) or nitrocellulose (NC) membranes (all others). Protein transfer was confirmed by Ponceau staining. Membranes were then blocked with either 5% milk + 0.1% Tween 20 in TBS (PVDF) or Odyssey blocking bu ffer (NC) (LI COR Biosciences, Lincoln, NE) for one hour. The membranes were then washed three times with 0.1% Tween 20 in TBS and incubated at 4C overnight in primary antibodies diluted with either 5% BSA + 0.1% Tween20 in TBS (PVDF) or Odyssey blocking buffer (NC). Primary antibodies were applied for: talin (Sigma, St. Louis, MO), MAFbx (Santa Cruz, Santa Cruz, CA), phospho -FOXO3a (Cell Signaling, Danvers, MA), phospho-Akt (Santa Cruz, Santa Cruz, CA), total Akt (Santa Cruz, Santa Cruz, CA). Beta actin (Abcam; Cambridge, MA) was used as a loading control. After overnight incubation, membranes were washed again with 0.1% Tween 20 in TBS three times and then incubated for 35 minutes in secondary antibody diluted with either 0.1% Tween 20 in TBS (PVDF) or Odyssey blocking buffer (NC). The secondary antibodies were IR Dye conjugated secondaries detectable at wavelengths of 680 or 800 nm (LI -COR) and non -fluorescent anti rabbit IgG. Membranes were washed three times with 0.1% Tween 20 in TBS and then once with either EDL plus (PVDF) or

PAGE 33

33 TBS (NC) before being scanned and detected using either Kodak film processing (PVDF) or the Odyssey infrared imaging system (NC) (LI COR). Statistical Analysis Group sample size was determined with power analysis of our prel iminary data. Comparisons between groups were made by a 2 -way or 3 -way full -factorial ANOVA, and when appropriate, Tukeys HSD test was performed post -hoc. Significance was established at p < 0.05.

PAGE 34

34 Figure 3 1. Experiment 1 d esign Aim 1, Hypothesis 1A. The purpose was to test elevated intracellular Ca2+ induced by treatment with A23187 as a dose -dependent model of skeletal muscle atrophy stimulating calpain proteolysis of intermediate filaments and increasing proteasome activity.

PAGE 35

35 Figure 3 2. Experiment 2 d esign Aim 1, Hypothesis 1B. Purpose was to determine the effect of exogenous administration of NO on calpain proteoloysis and proteasome activity after 60 minutes of treatment with A23187.

PAGE 36

36 Figure 3 3. Experiment 3 d esign Aim 1, Hypothesis 1B. Purpose was to compare the effects of NO on calpain proteoloysis and proteasome activity with those of calpeptin, a known calpain inhibitor, after 60 minutes of treatment with A23187.

PAGE 37

3 7 Figure 3 4. Experiment 4 d esign A im 2, Hypothesis 2A. Purpose was to test if elevated intracellular Ca2+ by treatment with A23187 induces visible myotube atrophy after 2428 hours.

PAGE 38

38 Figure 3 5. Experiment 5 d esign Aim 2, Hypothesis 2B. Purpose was test the eff ects of NO myotube atrophy following 2448 hours of treatment with A23187.

PAGE 39

39 CHAPTER 4 RESULTS Effects of Treatment with A23187 and NO on Molecular Markers Specific aim 1 was to test that elevated intracellular Ca2+ levels induced by treatment with A23187 is a dose -dependent model of skeletal muscle atrophy stimulating calpain proteolysis of intermediate filaments and increasing molecular markers of proteasome activity and to determine whether or not exogenous NO can attenuate these effects. Talin In experi ment 1, the ratio of cleaved to total talin increased significantly after 60 minutes of treatment with 20 M A23187 (Figures 4 1). And, although increased talin cleavage at other doses did not meet statistical significance with n=3 and p < 0.05, there was an apparent trend toward progressively more talin cleavage with increasing dose of A23187 (Figure 4 1). No significant changes were seen in talin cleavage after 30 minutes of treatment. In experiment 2, talin cleavage significantly increased after 60 minutes of treatment with 10 M A23187 and with 10 M A23187 + L NMMA, a blocker of endogenous NO release (Figure 4 2). Pre conditioning with both 1 M PAPA -NO and 10 M PAPA -NO appeared to attenuate the increase in talin cleavage after treatment with 10 M A23187, as cleavage levels were reduced to near control (not statistically different from control at p < 0.05) (Figure 4 2). Talin cleavage also significantly increased after treatment with 20 M A23187 + 1 M PAPA -NO and with 20 M A23187 + L NMMA (Figu re 4 2). However, pre -conditioning with 10 M PAPA NO appeared to attenuate the increase in talin cleavage after treatment with 20 M A23187, as the cleavage level was similar to control and significantly lower than with 20 M A23187 + 1 M PAPA -NO and wi th 20 M A23187 + L NMMA (Figure 4 2). Talin cleavage was significantly reduced by

PAGE 40

40 treatment with calpeptin, a specific calpain inhibitor, but concurrent treatment with PAPA NO did not provide additional protection (Figure 4 3). Akt In experiment 1, the r atio of phosphorylated (active) Akt to total Akt protein appeared to increase after 60 minutes of treatment with 10 M and 20 M doses of A23187, but the increases did not meet statistical significance with n=3 and p < 0.05 (Figure 4 4). No changes were seen after 30 minutes of treatment with A23187 at any dose (Figure 4 4). In experiment 2, the ratio of phosphorylated (active) Akt to total Akt protein significantly increased after 60 minutes of treatment with 10 M A23187 + 1 M PAPA NO, 10 M A23187 + 10 M PAPA -NO, and 10 M A23187 + L -NMMA, but there were no differences among these groups (Figure 4 5). Groups treated with 20 M A23187 showed no significant differences in Akt ratio (Figure 4 5). The ratio of phosphorylated Akt to total Akt was not alt ered with calpeptin treatment for any treatment group (Figure 4 6). FOXO3a In experiment 1, phosphorylated (inactive, cytosolic) FOXO3a protein expression did not significantly change after either 30 or 60 minutes of treatment with various doses of A23187 (Figure 4 7). However, treatment with 20 M A23187 for 60 minutes did appear to slightly decrease the expression of phospho -FOXO3a (Figure 4 7). In experiment 2, treatment with 10 M A23187 alone and with 10 M A23187 + 1 M PAPA -NO significantly increas ed phospho FOXO3a expression, but no other treatment groups showed significant differences (Figure 4 8). Treatment with calpeptin did not affect phospho -FOXO3a expression except when combined with 10 M PAPA NO; calpeptin + 10 M PAPA NO resulted in signi ficantly lower expression of phospho-FOXO3a after 60 minutes of treatment with 20 M A23187 (Figure 49).

PAGE 41

41 MAFbx In experiment 1, MAFbx protein expression did not significantly change after either 30 or 60 minutes of treatment with various doses of A23187 ( Figure 4 10). In experiment 2, MAFbx protein expression did not significantly change with any combination of 10 M A23187 and PAPA NO (Figure 4 11). Treatment with 20 M A23187, 20 M A23187 + 1 M PAPA -NO, and 20 M A23187 + 10 M PAPA NO significantly decreased MAFbx expression as compared to control groups, but there were no differences among these groups (Figure 4 11). Treatment with either dose of A23187 + L NMMA appeared to possibly increase MAFbx expression as compared to A23187 alone, but these d ifferences did not reach statistical significance at p < 0.05 (Figure 4 11). MAFbx protein expression was not altered with calpeptin treatment for any treatment group (Figure 4 12). Effects of A23187 Treatment and NO on Myotube Atrophy Specific aim 2 was to test if elevated intracellular Ca2+ by treatment with A23187 induces myotube atrophy (loss of size and/or total protein) and whether or not exogenous NO administration results in decreased atrophy. Protein Degradation Muscle protein degradation increase d in L6 myotubes undergoing 24 hours of treatment with A23187 (Figure 4 13). Cells exposed to 5, 10, and 20 M A23187 exhibited significantly more protein degradation than control. Although the differences among these three groups did not meet statistica l significance with n=3 and p < 0.05, there was an apparent trend toward progressively greater protein degradation with increasing doses of A23187. Doses of 50 M and 100 M were also tested in preliminary experiments, but cell viability was much poorer a t these high levels, so the doses were discontinued and protein was not measured.

PAGE 42

42 When cells were treated with A23187 with and without DETA NO for 24 hours there were no significant differences in protein degradation betw e en groups treated with NO and th ose treated without NO (Figure 4 14). Cells treated with A23187 had significantly less protein than cells without A23187, as was observed in the previous experiment. A23187 had a significant main effect at p < 0.0001, but there was no significant effect of NO on protein degradation. Myotube Image Analysis Visual images were captured after 24 and 48 hours of treatment with A23187 (Figure 4 15), and myotube diameter, length, and area were measured. Preliminary data images from groups treated with 50 M A23187 were analyzed for additional comparison. After 24 hours, myotube diameter was significantly decreased with 10 M A23187 treatment (Figure 4 16A), myotube length was significantly decreased with 50 M A23187 treatment (Figure 4 16B), and myotube are a was significantly decreased with 50 M A23187 treatment (Figure 416C). No other significant differences in myotube size were observed after 24 hours. After 48 hours, no differences in myotube dia meter were observed (Figure 4 16 A), but myotube length s ignificantly decreased with both 10 M and 50 M A23187 as compared to both untreated control and with 24 ho urs with 1 M A23187 (Figure 4 16B). Also after 48 hours, myotube area was significantly decreased with 10 M 20 M and 50 M A23187 as compared to both untreated control and with 24 hours with 1 M A23187 (Figure 4 16C). Visual images were again captured after 24 hours of treatment with A23187 w ith and without DETA NO (Figure 4 17) Image capture was stopped and analysis was omitted for cells tr eated for 48 hours due to myotube deterioration. Images of cells treated for 24 hours were analyzed; myotube diameter, length, and area were measured (Figure s 4 18, 4 19, 4 20). Myotubes treated with 1 M DETA NO alone had siginificantly increased diamet er, length, and area when compared to untreated cells (Figure s 4 18, 4 19, 4 20).

PAGE 43

43 Treatment with eithe r 10 M A23187 or 20 M A23187 alone significantly decreased myotube diameter while concurrent NO treatment significantly attenuated the decreases (Fig ure 4 18). For cells treated with 10 M A23187, those without DETA -NO had significantly decreased diameter as compared to untreated controls. However, concurrent treatment with either 1 M DETA NO or 10 M DETA NO resulted in myotube diameter similar to that of untreated control cells. Additionally, c oncurrent treatment with 10 M DETA NO signi fi cantly increased myotube diameter as compared to 10 M A23187 alone For cells treated with 20 M A23187, those without DETA NO had significantly decreased dia meter as compared to untreated controls. However, c oncurrent treatment with either 1 M DETA NO or 10 M DETA -NO resulted in significantly greater myotube diameter than the lower dose 10 M A23187 alone, indicating partial protection of myotube diame ter. Concurrent treatment with 1 M DETA NO also significantly increased myotube diameter as compared to 20 M A23187 alone. Treatment with either 10 M or 20 M A23187 alone significantly decreased myotube length, but NO had no effect on the decreases in le ngth for any dose combination (Figure 4 19). There was a significant main effect for DETA NO treatment, but increased length was only NO alone. DETA NO failed to attenuate the decreased length seen in groups treated with A23187. T reatment with either 10 M or 20 M A23187 alone also significantly decreased myotube area, but concurrent NO treatment did attenuate the loss of myotube area for cells treated at the lower dose of 10 M A23187 (Figure 4 20). For cells treated with 10 M A23187, those without DETA NO and those with 1 NO had significantly decreased myotube area as compared to untreated controls. However, cells concurrently treated with 10 M DETA NO had myotube area similar to untreated controls and significantly greater than those

PAGE 44

44 with 10 M A23187 alone. F NO significantly changed the myotube area.

PAGE 45

45 Figure 4 1. Talin cleavage in L6 myotubes after 60 minutes of treatment with A23187. Values represent the mean SEM (n=3). *Significa ntly different from control (p < 0.05).

PAGE 46

46 Figure 4 2. Talin cleavage in L6 myotubes after 60 minutes of treatment with A23187 and/or PAPA NO or L NMMA pre -conditioning. Values represent the mean SEM (n=3). *Significantly different from contr ol (p < 0.05). #Significantly different from 20 M A23187 + 1 M NO and 20 M A23187 + L NMMA (p < 0.05).

PAGE 47

47 Figure 4 3. Talin cleavage in L6 myotubes after 60 minutes of treatment with 20 M A23187 and/or PAPA NO pre -conditioning with and withou t calpeptin. Values represent the mean SEM (n=4). *Significantly different from 20 M A23187 control (p < 0.05). #Significantly different from same treatment without calpeptin (p < 0.05).

PAGE 48

48 Figure 4 4. Phopho/Total Akt protein expres sion in L6 myotubes after 30 or 60 minutes of treatment with A23187. Values represent the mean SEM (n=3).

PAGE 49

49 Figure 4 5. Phospho/Total Akt protein expression in L6 myotubes after 60 minutes of treatment with A23187 and/or PAPA NO or L NMMA pr e -conditioning. Values represent the mean SEM (n=3). *Significantly from control groups (p < 0.05).

PAGE 50

50 Figure 4 6. Phospho/Total Akt protein expression in L6 myotubes after 60 minutes of treatment with 20 M A23187 and/or PAPA NO pre -conditioni ng with and without calpeptin. Values represent the mean SEM (n=4).

PAGE 51

51 Figure 4 7. PhosphoFOXO3a protein expression in L6 myotubes after 30 or 60 minutes of treatment with A23187. Values represent the mean SEM (n=3).

PAGE 52

52 Figure 4 8 PhosphoFOXO3a protein expression in L6 myotubes after 60 minutes of treatment with A23187 and/or PAPA NO or L NMMA pre -conditioning. Values represent the mean SEM (n=3). *Significantly different from control groups (p < 0.05).

PAGE 53

53 Figure 4 9. PhosphoFOXO3a protein expression in L6 myotubes after 60 minutes of treatment with 20 M A23187 and/or PAPA NO pre -conditioning with and without calpeptin. Values represent the mean SEM (n=4). *Significantly different from control (p < 0.05).

PAGE 54

54 Figure 4 10. MAFbx protein expression in L6 myotubes after 30 or 60 minutes of treatment with A23187. Values represent the mean SEM (n=3).

PAGE 55

55 Figure 4 11. MAFbx protein expression in L6 myotubes after 60 minutes of treatment with A23187 and/or PAPA -NO or L -NMMA pre -conditioning. Values represent the mean SEM (n=3). *Significantly different from control groups (p < 0.05).

PAGE 56

56 Figure 4 12. MAFbx protein expression in L6 myotubes after 60 minutes of treatment with 20 M A23 187 and/or PAPA NO pre -conditioning with and without calpeptin. Values represent the mean SEM (n=4).

PAGE 57

57 Figure 4 13. Total protein concentrations for L6 myotubes after 24 hours treatment with A23187. Values represent the mean SEM (n=3). *Signif icantly different from control ( p < 0 .05).

PAGE 58

58 Figure 4 14. Total protein concentrations for L6 myotubes after 24 hours treatment with A23187 and/or DETA NO. Values represent the mean SEM (n=3). *Significantly different from all control groups (p < 0 .05). Significant main effect of A23187 treatment ( p < 0 .0001).

PAGE 59

59 Figure 4 15. Representative images of L6 myotubes after 24 and 48 hours treatment with A23187. Left column represents 24 hours treatment. Right column r epresents 48 hours treatment. Top row represents control groups (DMSO only). Second row represents groups treated with 1 M A23187. Third row represents groups treated with 10 M A23187. Fourth row represents groups treated with 20 M A23187. 24 hr CON 48 hr CON

PAGE 60

60 F igure 4 16. Image analysis of L6 myotubes after 24 and 48 hours treatment with A23187. A) Average myotube diameter B) Average myotube length. C) Average myotube area. Values represent the mean SEM (n= 7 10). *Significantly different from untreated cont rol. #Significantly different from 24 hours of 1 M A23187. A B C

PAGE 61

61 Figure 4 17. Representative images of L6 myotubes after 24 hours treatment with A23187 and/or DETA NO. Left column represents control cells no A23187. Middle cells CON CON 1 CON 1 1 10 0 1 10

PAGE 62

62 Figure 4 1 8 Image analysis of L6 myotube diameter after 24 hours treatment with A23187 and/or DETA NO. V alues represent the mean SE M (n= 2842). *Significantly different from control at 0 M NO ( p < 0 .05). #Significantly different from control at 1 M NO ( p < 0 .05). Significantly different from control at 10 M NO ( p < 0 .05). Significantly different from 10 M A23187 + 0 M NO ( p < 0 .05). Significantly p < 0 .05). Significant main effect of A23187 treatment ( p < 0 .0001). Significant main effect of NO treatment ( p < 0 .0001).

PAGE 63

63 Figure 4 1 9. Image analysis of L6 myotube length after 24 hours treatment with A23187 and/or DETA NO. Values represent the mean SEM (n =28 42). *Significantly different from all controls ( p < 0 .05). #Significantly different from control at 0 M NO ( p < 0 .05). Significant main effect of A23187 treatment ( p < 0 .0001). Significant main effect of NO treatment (p=0.0008). Significant interac tion between A23187 and NO (p=0.02).

PAGE 64

64 Figure 4 20. Image analysis of L6 myotube area after 24 hours treatment with A23187 and/or DETA NO. Values represent the mean SE M (n=28 42). *Significantly different from control at 0 M NO ( p < 0 .05). #Signific antly different from control at 1 M NO ( p < 0 .05). Significantly different from control at 10 M NO ( p < 0 .05). Significantly different from 10 M A23187 + 0 M NO ( p < 0 .05). Significantly different from 1 1 p < 0 .05). Significant main effect of A23187 treatment ( p < 0 .0001). Significant main effect of NO treatment ( p < 0 .0001). Significant interaction between A23187 and NO (p=0.0009).

PAGE 65

65 CHAPTER 5 DISCUSSION Main Findings This study demonstrated the dose -dependent effects of NO on calpain proteolysis of intermediate f ilaments in skeletal muscle myotubes during a Ca2+ challenge connected the changes in molecular markers of calpain proteolysis and proteasome activity with myotube atrophy (loss of size and/or total protein) during a Ca2+ challenge and exogenous NO adminis tration. Treatment with A23187 significantly increased the cleavage of talin, a protein known to be degraded by calpains, while treatment with PAPA NO had a significant protective effect that varied with the doses of A23187 and PAPA NO administered. The effects of A23187 on talin cleavage and the protective effects of PAPA -NO were independent of other molecular markers of proteolysis. Also, treatment with A23187 caused significant loss of cellular protein and myotube size while DETA NO prevented loss o f myotube size during treatment with A23187 at certain dose combinations These data demonstrate that NO can protect against calpain proteolysis and calpain -induced myotube atrophy during a Ca2+ challenge in L6 myotubes. A23187 Causes Degradation of Inter mediate Filaments by Calpain s Independent of Proteasome Activity We hypothesized that elevation of intracellular Ca2+ by treatment with A23187 would cause increased proteolysis of intermediate filaments (e.g. talin) by calpain and increased markers of prot easome activation (e.g. FOXO3a, MAFbx). We found that treatment with A23187 does cause increased proteolysis of the intermediate filaments by calpain s but this effect appears to be independen t of proteasome activ ation Calpains are known to cleave the in termediate filament talin, leaving a 190 -kDa fragment that is distinguishable by Western blotting fro m the 235kDa intact protein (19 ). Treatment with

PAGE 66

66 A23187 significantly increased calpain proteolysis of intermediate filaments, as measured by ratio of cleaved/intact talin protein (Figure 4 1). This effect was dose -depend ent, as incrementally increasing A23187 was associated with an apparent, yet not always statistically significant, incremental increase in talin cleavage (Figure 4 1). Treatment with 10 M and 20 M A23187 caus ed statistically significant increase s in cleavage (Figure 4 1 4 2 ). Our results are consistent with evidence from C2C12 myoblasts that A23187 increases talin proteolysis (19 ). Although in our first experiment the increase in cle avage was not statistically significant with 10 M A23187 (Figure 4 1), w e expect ed that with a higher number of samples it would be. T herefore we continued experiments with both 10 M A23187 and 20 M A23187, and in our second experiment cells treated wi th 10 M A23187 did show more cleavage than controls (Figure 4 2). Talin cleavage is an indirect measure of calpain activity that has been accepted in the literature as a mea sure of calpain proteoloysis (19, 42). However, in order to test that the talin c leavage was caused by calpai n s, we administered calpastatin, a known specific inhibitor of calpain s I and II, to cells treated with 20 M A23187 (with and without NO). With calpastatin blocking calpain activity, talin cleavage dropped dramatically for a ll groups (Figure 4 3), which suggests that the talin cleavage seen in response to A23187 treatment is in fact the result of calpain proteolysis. Although we saw significant increases in calpain proteolysis, we did not observe concurrent changes in markers of proteasome act ivity in response to A23187 treatment. We chose to measure markers of proteasome activity in an attempt to connect calpain proteolysis of intermediate filaments with overall proteasome degradation. Previous research has demonstrated tha t cleavage of intermediate filaments and myofibrillar release by calpains precedes degradation by the proteasome (14, 27, 36). Recent research has also suggested that calpain activity is

PAGE 67

67 necessary for proteasome action in t he rat diaphragm (31). We measu red phospho-Akt/total Akt, phospho-FOXO3a, and MAFbx protein expression for the three experiments designed to test specific aim 1. Increased Akt activation via phosphorylation by PI3K has been shown to be sufficient alone to downregulate the acitivty of proteaso me E3 ligases MAFbx and MuRF1 (4, 29). However, phospho-Akt suppression of MAFbx and MuRF1 expression is dependent up on FOXO transcription factors (4, 29, 33). Unphosphorylated FOXO proteins are active and locate to the nucleus where they can stim ulate E3 ligase transcription ( 40). Phospho-Akt inactivates FOXO proteins (including FOXO3a) by phosphorylation, which causes them to be translocated away from the nucleus and into the cytosol, where phospho-FOXO proteins are targeted by the proteasome fo r degradation (25, 40). Thus, if A23187 were causing proteasome a ctivity, we would expect to see decreases in both phospho-Akt and phosphoFOXO3a expression and an increase in MAFbx expression However, we did not see any significant changes in these pro teins with A 23187 treatment alone and treat ment with calpeptin (blocking calpains) had no significant effects (Figures 4 4, 46, 4 7, 4 9, 4 10, 412). While the lack of significant increase in any of these proteins following A23187 treat ment was surprising, there are a few possible explanations. First, it is possible that proteasome activity and changes in these proteins were missed at 60 minutes. We chose to harvest cellular protein after 60 minutes of incubation with A23187 based upon previous evidence that proteasome activity in L6 myotubes increases most rapidly during the first 40 minutes of incubation with A23187 and then continues to increase significantly for 120 minut es (21 ). Importantly, however, this study did not measure proteins involved i n cellular signaling but rather overall proteasome activity by measuring degradation of a fluorescent substrate (21 ). Based on the little previous data that we had, w e believed that 60 minutes would be sufficient time to see proteasome activity

PAGE 68

68 but still brief enough not to miss evidence of the calpain cleavage that should precede it. Incubating for a longer period would have risked losing the cleaved talin protein to proteasome degradation, and since we were primarily concerned with calpain activity in t his study, we chose to limit the time to 60 minutes. It is possible, then that proteasome activity was occurring but that the the molecular markers that we measured were not captured at 60 minutes. Second, it is possible that proteasome activity was occ urring but not via the inactive Akt/FOXO/E3 ligase pathway. These proteins, while associated with skeletal muscle atrophy, are only a few of the myriad of indirect measurements of proteolytic activity. We chose to study this pathway based on previous wor k that has shown calpain activity inhibiting Akt signaling in skeletal muscle (31 ) and previous data from our laboratory that has shown a significant relationship between Akt signal ing and NO in skeletal muscle (7 ). We chose to measure MAFbx as a represen tative of E3 ligases because of its predominance in the literature and availability of a reliable antibody. However, it is possible that other E3 ligases, such as MuRF1, would have yielded a different result. Third, it is possible that in L6 myotubes, pro teolysis by calpains and protesomes have a different relationship than in other skeletal muscle cell lines. L6 myotubes differentiate into mature myotubes with striations, but they do not have functional sarcomeres and do not contract. We chose this cell line because other cell lines, such as C2C12s, would not withstand the Ca2+ challenge as mature myotubes because they would contract, dislodge from the plate, and die. We attempted to use both cell lines in initial experiments to confirm that C2C12 myot ubes were not a viable choice for this study, and this is likely why the only previous study of the effects of A23187 and NO on skeletal muscle cells in culture was done in myoblasts (19 ). While L6 myotubes do not have functional sarcomeres, they do expre ss all proteins that we studied, as

PAGE 69

69 evidenced by our data. It is still possible, however, that the sequence of proteolytic events (requisite calpain cleavage followed by proteasome action) is different for this cell line. The one study previously mention ed that measured proteasome activity in response to A23187 did not measure any markers of calpain activity (21 ). Therefore, it is possible that calpain and proteasome activity do not occur in the same sequence or with the same timing in L6 myotubes as in other cell lines. Considerably more research would need to be done on this subject before assessing how likely this possibility is, however, it would explain the consistency of our results showing calpain activity changes without concomitant changes in th e other atrophy markers. Exogenous NO Prevents Degradation of Intermediate Filaments by Calpain s Independent of Proteasome Activity We hypothesized that exogenous NO administration would attenuate calpain proteoloysis and proteasome activity after treatmen t with A23187. We found that NO does protect against proteolysis of the intermediate filaments by calpains, but this effect appears to be independent of proteasome activity and is dose -dependent. NO has been proposed as a potential regulator of calpain activity in various tissues by a sma ll number of previous studies (5, 19, 22, 42). We used two doses of PAPANO (1 M and 10 M ) crossed with the two doses of A23187 that appeared to induce cleavage of the intermediate filaments by calpains (10 M and 20 M ) to test the ability of NO to protect against calpains during a Ca2+ challenge. We also used L NMMA, an inhibitor of endogenous NO release, to further assess the role of NO. Both 1 M and 10 M PAPA -NO were able to significantly attenuate talin cleava ge by calpains after treatment with 10 M A23187 (Figure 4 2). However, only 10 M PAPA NO was able to attenuate talin cleavage after treatment with 20 M A23187 (Figure 4 2). Treatment with L NMMA resulted in increased talin cleavage as compared to untr eated controls but did not have a significantly different effect than treatment

PAGE 70

70 with A23187 alone (Figure 4 2). These results suggest that NO does mediate calpain activity during a Ca2+ challenge, but that the dose of NO required to protect intermediate f ilament proteins from degradation is dependent upon the level of Ca2+. Since NO can have either positive or negative effects throughout the cell depending on the dose, it is important to note that a dose as low as 1 M PAPA -NO had a significant protective effect and that 10 M PAPA -NO protected at both doses of A23187 but was not required for protection at the relatively lower dose of A23187. And while treatment with L NMMA did not produce an increase in talin cleavage at a level that reached statistical significance at n=6 and p < 0.05, it appears that blocking endoge nous NO with L NMMA could cause a slight increase (Figure 4 2). Because this study was primarily concerned with exogenous NO as a potential mediator, we did not study L NMMA or other NO bloc kers alone. However, future experiments exploring the the effect of endogenous NO on calpain proteolysis of intermediate filaments would be useful in determining if endogenous NO could protect skeletal muscle from calpain proteolysis or if exogenous NO is required in therapeutic doses as studied here. One previous study has examined endogenous NO release on calpain activity in a mechanical strain model with positive results (42). Finally, we showed that NO did not attenuate calpain activity as significan tly and universally as calpeptin and that NO + calpeptin treatment was not different from treatment with calpeptin alone (Figure 4 3). This is likely due to the potency of the calpeptin and its efficacy as a blocker of both calpain I and calpain II. Altho ugh NO had protective effects on the intermediate filaments by inhibiting proteolysis by calpains, treatment with NO had few significant effects on markers of proteasome a ctivation If NO was protecting against proteasome activity, we would expect to see increased Akt

PAGE 71

71 phosphorylation and phospho -FOXO3a expression and decreased MAFbx expression. However, our data did not support this. Akt phosphorylation increased with treat ment with 10 M A23187 + all dose s of NO or L NMMA, but there were no differences among these groups (Figure 4 5). Thus, NO did not affect Akt phosphorylation in our study. This somewhat conflicts with evidence published by our lab that shows eNOS knockout mice treated with 0.4 M A23187 have dramatically reduced Akt phosphorylation, which is resto red by PAPA NO administration (7 ). However, this study used very low levels of A23187 and was conducted in a different model, so comparison is difficult. Since Akt phosphorylation did increase with 10 M A23187 but not with any of the treat ment groups at the higher dose of A23187, i t may be that the effect of A23187 on Akt phosphorylation is dose and/or time dependent. Dose -dependence is seen in many different physiological molecules, including NO, so it is not unrealistic to consider that Akt phosphorylation could be triggered by certain levels of intracellular Ca2+ but could be unchanged (as in our study) or even reversed by other levels. Phospo-FOXO3a expression paradoxically increased with 10 M A23187 alone and with 1 M PAPA -NO but di d not change in any other group (Figu re 4 8). This result is difficult to explain, and in light of the lack of significant changes in phospho-FOXO3a throughout our study (Figures 4 7, 48 4 9) it may be that this protein is not affected by A23187, calpai n activity, or NO in L6 myotubes. And, as previously stated, it is possible that calpain and proteasome activity do not occur in the same sequence or with the same timing in L6 myotubes as they do in other cell lines. MAFbx expression paradoxically decrea sed with 20 M A23187 alone and with 20 M A23187 + either dose of PAPA NO (Figure 4 11). No other significant changes in MAFbx were

PAGE 72

72 seen throughout our study (Figures 4 10, 411, 4 12). Again, this result is difficult to explain, and it may be that this protein is not affected by A23187, calpain activity, or NO in L6 myotubes. And, as previously stated, it is possible that calpain and proteasome activity do not occur in the same sequence or with the same timing in L6 myotubes as they do in other cell li nes. Although there were few significant differences in Akt phosphorylation, phosphoFOXO3a, or MAFbx expression with A23187 and NO treatments, it should be noted that the lack of changes is remarkably consistent, and that we would expect this to be the ca se if this pathway was either not captured at the 60 minute treatment end or not active in our L6 myotubes in this atrophy model. Since we expected these proteins to be expressed together and to work in concert, relative consistency in their lack of change among treatment conditions is not especially surprising and further supports the idea that proteasome activity was either missed by our 60 minute treatment or that the coupling of calpain and proteasome activity is not the same in L6 myotubes as it is in other cells. High Doses of A23187 C ause L6 Myotube A trophy We hypothesized that elevation of intracellular Ca2+ by treatment with A23187 causes myotube atrophy. This is the first study to our knowledge to attempt to connect the markers of calpain prote olysis with myotube atrophy and we found that the same doses of A23187 that increase calpain activity also cause cause myotube atrophy over 2448 hours After 24 hours of treatment, 5 M 10 M and 20 M doses of A23187 caused loss of total protein (Fig ure 4 13). This is particularly important in light of our data showing that proteolysis by calpains is increased with 60 minutes treatment with 10 M and 20 M A23187 (Figures 4 1, 4 2). After 24 hours of treatment, there was little change in myotube dia meter, length, or area with any dose of A23187 below 50 M (Figure 4 15). However, after 48 hours, cells treated with 10 M and 20 M A23187 (as well as 50 M ) showed decreased myotube area (Figure 415). The loss of

PAGE 73

73 protein by 24 hours and loss of myotube area by 48 hours are evidence of A23187induced myotube atrophy. Furthermore, we can connect the increase in calpain proteolysis of the intermediate filaments seen in the first 60 minutes of treatment (Figure 4 1) with this evidence of atrophy at the same doses (Figures 4 13 and 4 15) over 24 and 48 hours and conclude that the calpain proteolysis induced by A23187 is a sign of atrophy, not simply cellular remodeling. Exogenous NO Protects L6 M yotube s from A23187-Induced A trophy We hypothesized that exo genous NO would attenutate myotube atrophy after treatment with A23187, and we found that while it did not attenuate the loss of total protein associated with A23187 treatment, exogenous NO did attenuate loss of myotube size at certain dose combinations When cells were treated with DETA -NO along with A23187, protein degradation decreased with A23187 treatment but was not affected by NO after 24 hours (Figure 4 14). The lack of protection by NO was not expected, especially in light of the preservation of myotube size discussed below. It is possible that NO does not protect against total protein loss during a Ca2+ challenge. However, a significant number of cells were lost during the process of stopping treatment, washing with PBS, image capturing, and h arvesting. This likely affected the results of this experiment and may have contributed to the lack of significant results. Although it did not preserve total cellular protein, concurrent treatment with NO did preserve myotube size. For c ells treated wit h 10 M A23187, both 1 M and 10 M DETA NO preserved myotube diameter to the level of untreated controls; 10 M DETA NO also significantly increased myotube diameter as compared to cells treated with 10 M A23187 alone. Additionally, 10 M DETA NO preser ved myotube area to a level similar to untreated controls; myotube area for these cells was significantly greater than for those treated with 10 M A23187 alone. For cells treated with 20 M A23187, 1 M DETA -NO significantly increased myotube diameter as compared to 20 M A23187 alone and both 1 M DETA NO or 10 M

PAGE 74

74 DETA NO resulted in significantly greater myotube diameter than the lower dose 10 M A23187 alone, indicating partial protection of myotube diame ter. However, NO had no protective effects on myotube area during the higher dose A23187 treatment These effects on myotube size indicate that NO can protect myotubes from loss of size but that the effects are dependent upon the severity of the Ca2+ challenge. We conclude that NO protect s L6 myotube s from A23187induced atrophy, as measured by changes in myotube size, but that the dose of NO required to protect the myotubes is dependent up on the dose of A23187 and that the deleterious effects of higher levels of intracellular Ca2+ may be too much for NO to counteract. These results agree with our molecular markers results, which showed that NO protects against calpain cleavage of intermediate filaments at the same doses of A23187 and NO. Thus, we conclude that the attenuation of calpain cleavage of intermediate filaments is evidence of myotube atrophy, not simply cellular remodeling and that NO can protect L6 myotubes from calpain-induced atrophy. Limitations and Future Directions This study was limited by several factors, the first of which is the u se of an ionophore Using A23187 as our independent variable to raise intracellular Ca2+ is not a physiological stimulus of Ca2+ influx. A23187 is an ionophore that allows divalent cations, such as Ca2+ to cross the cell membrane and raise intracellular concentrations. Although this ionophore is widely used in cell culture as a method of increasing intracellular Ca2+, it does have other effects on the cell, such as inhibiting oxidative phosphorylation and acting as an antibiotic. We do not believe that the secondary effects of the ionophore significantly compromise our study results because the effects should not impact atrophy or associated signaling in non -contracting cells. Second, this study was limited by the use of indirect measures of proteolysis. Talin cleavage was used as an indirect marker of calpain activity. However, this is not uncommon in

PAGE 75

75 the literature, and our results showing calpeptin reversing the talin cleavage support the assumption that talin cleavage is representative of calpain ac tivity. Akt (de -)phosporylation is limited because alone it would be a very indirect indicator of proteolytic activity. However, we also measured two of the proteins downstream of Akt: FOXO and MAFbx. Using phosphoFOXO3a was somewhat indirect as well b ecause we assume that loss of phospho-FOXO3a represents de -phosphorylation and translocation to the nucleus. However, we also measured MAFbx, the transcription of which is upregulated by FOXO3a. The measurement of only MAFbx expression to represe nt E3 li gases is also limiting. Future studies should isolate nuclear and cytosolic fractions to measure FOXO3a/FO XO3a~P and should also measure MuRF1 expression. Third, this study was limited in that we could only examine certain timepoints as snapshots of wha t was occurring in the myotubes. It is highly possible that some changes in proteins measured were missed because they were either degraded during the 60 minute treatment period or not yet activated or elevated. A low number of samples also compromised o ur ability to elucidate the cell signaling occurring in our study. Future studies should examine different time points and increase the number of samples taken. Finally, this study is limited as are all in vitro cell culture studies by our inability to ap ply these results directly to a whole organism. However, in vitro studies such as this one are necessary to study the mechanisms behind clinical problems like skeletal muscle atrophy with maximal experimental control and minimal influence of other physiol ogical factors. Conclusions We conclude that elevation of intracellular Ca2+ by treatment with A23187 causes increased proteolysis of intermediate filaments (e.g. talin) by calpain independent of proteasome activity and that exogenous NO administration att enuates calpain proteoloysis but not

PAGE 76

76 proteasome activity after treatment with A23187. We further conclude that the increases in calpain activity induced by treatment with A23187 cause s myotube atrophy and that exogenous NO protects L6 myotubes from A23187induced atrophy.

PAGE 77

77 LIST OF REFERENCES 1. Bartoli M and Richard I. Calpains in muscle wasting. International Journal of Biochemistry and Cell Biology 37: 21152133, 2005. 2. Bechet D, Tassa A, Taillandier D, Combaret L, Attaix D. Lys osomal proteolysis in skeletal muscle. International Journal of Biochemistry and Cell Biology 37: 20982114, 2005. 3 Bergamini E. Autophagy: A cell r epair mechanism that retards ag ing and age associated diseases and can be intensified pharmacologically. M ol ecular Aspects of Med icine 2 7: 403410, 2006. 4 Bodine SC, Latres E, Baumhueter S, Lai VKM, Nunez L, Clarke B, Poueymirou WT, Panaro FJ, Na E, Dharmarajan K, Zhen Qiang P, Valenzuela DM, DeChiara TM, Stitt TN, Yancopoulos GD, Glass DJ. Identification o f Ubiquitin Ligases Required for Skeletal Muscle Atrophy. Science 294: 17041708, 2001. 5 Chohan PK, Singh RB, Dhalla NS, and Netticadan T. L arginine administration recovers sarcoplasmic reticulum function in ischemic reperfused hearts by preventing calp ain activation. Cardiovascular Research 69: 152163, 2006. 6 Costelli P, Reffo P, Penna F, Autelli R, Bonelli G, Baccino FM. Ca2+dependent proteolysis in muscle wasting. International Journal of Biochemistry and Cell Biology 37: 21342146, 2005. 7 Dren ning JA, Lira VA, Soltow QA, Cannon CN, Valera LM, Brown DL, Criswell DS. Endothelial nitric oxide synthase is involved in calcium induced Akt signaling in mouse skeletal muscle. Nitric Oxide In Press, 2009. 8 Du J, Wang X, Miereles C, Bailey JL, Debigare R, Zheng B, Price SR, and Mitch WE. Activation of caspase 3 is an initial step triggering accelerated muscle proteolysis in catabolic conditions. Journal of Clinical Investigations 113: 115123, 2004. 9 Furano K & Goldberg AL. The activation of protein degradation in muscle by Ca2+ or muscle injury does not involve a lysomsomal mechanism. The Biochemical Journal 237: 859864, 1986. 10. Gissel H. The role of Ca2+ in muscle cell damage. Annals of the New York Academy of Sciences 1066: 166180, 2005. 11. Gi ssel H and Clausen T. Excitation induced Ca2+ influx and skeletal muscle cell damage. Acta Physiologica Scandinavi c a 171: 327334, 2001. 12. Glass DJ. Molecular mechanisms modulating muscle mass. Trends in Molecular Medicine 9: 344350, 2003.

PAGE 78

78 13. Glass DJ. Skeletal muscle hypertrophy and atrophy signaling pathways. International Journal of Biochemistry and Cell Biology 37: 19741984, 2005. 14. Goll DE, Thompson VF, Li H, Wei W, and Cong J. The calpain system. Physiological Reviews 83: 731801, 2003. 15. Go mes MD, Lecker SH, Jagoe RT, Navon A, Goldberg AL. Atrogin 1, a muscle specific F -box protein highly expressed during muscle atrophy. Proceedings of the National Academy of Sciences 98: 1444014445, 2001. 16. Grune T, Merker K, Sandig G, and Davies KJ. Sel ective degradation of oxidatively modified substrates by the proteasome. Biochemical and Biophysical Research Communications 305: 709718, 2003. 17. Hershko A, Ciechanover A. The ubiquitin system. Annual Reviews in Biochemistry 67: 425427, 1988. 18. Jackm an RW and Kandarian SC. The molecular basis of skeletal muscle atrophy. American Journal of Physiology Cell Physiology 287: C834C843, 2004. 19. Koh TJ and Tidball JG. Nitric oxide inhibits calpain -mediated proteolysis of talin in skeletal muscle cells. A merican Journal of Physiology Cell Physiology 279: C806C812, 2000. 20. Li YP, Chen Y, Li AS, and Reid MB. Hydrogen peroxide stimulates ubiquitin conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes. Amer ican Journal of Physiology Cell Physiology 285: C806812, 2003. 21. Menconi MJ, Wei W, Yang H, Wray CJ, Hasselgren PO. Treatment of cultured myotubes with the calcium ionophore A23187 increase s proteasome activity via a CaMK II-caspase -calpain -dependent me chanism. Surgery 136: 135142, 2004. 22. Michetti M, Salamino F, Melloni E, and Pontremoli S. Reversible inactivation of calpain isoforms by nitric oxide. Biochemical and Biophysical Research Communications 207: 10091014, 1995. 23. Musial A and Elissa NT. Inducible nitric -oxide synthase is regulated by the proteasome degradation pathway. Journal of Biological Chemistry 276: 2426824273, 2001. 24. Otani K, Han D, Ford E, Garcia -Roves PM, Ye H, Horikawa Y, Bell GI, Holloszy JO, and Polonsky KS. Calpain syste m regulates muscle mass and glucose transporter GLUT4 turnover. Journal of Biological Chemistry 279: 2091520920, 2004. 25. Plas DR, Thompson CB. Akt activation promotes degradation of tuberin and FOXO3a via the proteasome. Journal of Biological Chemistry 278: 1236112366, 2003.

PAGE 79

79 26. Powers SK, Kavazis AN, and DeRuisseau KC. Mechanisms of disuse muscle atrophy: role of oxidative stress. American Journal of Physiology Regulatory, Integrative, and Comparative Physiology 288: 337344, 2005. 27. Powers SK, Kavaz is AN, & McClung JM. Oxidative stress and disuse muscle atrophy. Journal of Applied Physiology 102: 238997, 2007. 28. Sacheck JM, Ohtsuka A, McLary SC and Goldberg AL. IGF 1 stimulates muscle growth by suppressing protein breakdown and expression of atrop hy -related ubiquitin ligases, atrogin 1 and MuRF1. Am erican J ournal of Physiol ogy Endocrinol ogy and Metab olism 287: E591601, 2004. 29. Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, Walsh K, Schiaffino S, Lecker SH and Goldberg AL. FOXO tra nscription factors induce the atrophyrelated ubiquitin ligase atrogin 1 and cause skeletal muscle atrophy. Cell 117: 399-412, 2004. 30. Senf SM, Dodd SL, McClung JM and Judge AR. Hsp70 overexpression inhibits NF {kappa}b and FOXO 3a transcriptional activities and prevents skeletal muscle atrophy. The FASEB Journal 22: 383645, 2008. 31. Smith IJ and Dodd SL. Calpain activation causes a proteosome dependent increase in protein degradation and inhibits the Akt signaling pathway in rat diaphragm muscle. Experi mental Physiology 92:56173, 2007. 32. Spencer MJ, Lu B, and Tidball JG. Calpain II expression is increased by changes in mechanical loading of muscle in vivo. Journal of Cellular Biochemistry 64: 5566, 1997. 33. Stitt TN, Drujan D, Clarke BA, Panaro F, T imofeyva Y, Kline WO, Gonzalez M, Yancopoulos GD and Glass DJ. The IGF 1/PI3K/Akt pathway prevents expression of muscle atrophy induced ubiquitin ligases by inhibiting foxo transcription factors. Molecular Cell 14: 395403, 2004. 34. Taillander D, Aurousse au E, Meynial -Davis D, Bechet D, Ferrara M, Cottin P, Ducastataing A, Bigard X, Guezennec CY, Schmid HP, and Attaix D. Coordinate activation of lysosomal, Ca2+activated and ATP ubiquitin -dependent proteases in the unweighted rat soleus muscle. The Biochem ical Journal 316: 6572, 1996. 35. Taillander D, Combaret L, Pouch MN, Samuels SE, Bechet D, Attaix D. The role of ubiquitin -proteasome -dependent proteolysis in the remodeling of skeletal muscle. Proceedings of the Nutrition Society 63: 357361, 2004. 36. Tidball JG and Spencer MJ. Expression of a calpastatin transgene slows muscle wasting and obviates changes in myosin isoform expression during murine muscle disuse. Journal of Physiology 545: 819828, 2002. 37. Tischler ME, Rosenberg S, Satarug S, Henrikse n EJ, Kirby CR, Tome M, and Chase P. Different mechanisms of increased proteolysis in atrophy induced by denervation or unweighting of rat soleus muscle. Metabolism 39: 756763, 1990.

PAGE 80

80 38. Thompson VF, Lawson K, and Goll DE. Effect of -calpain on m -calpain Biochemical and Biophysical Research Communications 267: 495499, 2000. 39. Vassilakopoulos T, Deckman G, Kebbewar M, Rallis G, Harfouche R, and Hussain SNA. Regulation of nitric oxide production in limb and ventilatory muscles during chronic exercise tr aining. American Journal of Physiology Lung Cell Molecular Physiology 284: L452L457, 2002. 40. Van der Heide LP, Hoekman MF, Smidt MP. The ins and outs of FOXO shuttling: mechanisms of FOXO translocation and transcriptional regulation. The Biochemical Journal 380: 297 309, 2004. 41. Wray CJ, Mammen JM, Hershko DD, Hasselgren PO. Sepsis upregulates the gene expression of multiple ubiquitin ligases in skeletal muscle. International Journal of Biochemistry & Cell Biology 35: 698705, 2003. 42. Zhang JS, Kraus WE, and Truskey GA. Stretch -induced nitric oxide modulates mechanical properties of skeletal muscle cells. American Journal of Physiology Cell Physiology 287: 292299, 2004.

PAGE 81

81 BIOGRAPHICAL SKETCH Elizabeth Henderson Zeanah is the daugh ter of William Ross Zeanah, M.D. and Sharon Cook Zeanah. Ms. Zeanah was born in Houston, Texas and raised in Gainesville, Florida, where she attended Eastside High School and graduated from the International Baccalaureate (IB) program. Ms. Zeanah then mo ved to Los Angeles, California, where she received a Bachelor of Science in Kinesiology from the University of Southern California (USC) and a minor in Public Health from the USC Keck School of Medicine in 2006. While at USC, Ms. Zeanah assisted with proj ects in glucose transport and lipid metabolism in skeletal muscle under the direction of Dr. Lorraine Turcotte. She was also a three year letterwinner, PAC 10 champion, and NCAA Championships finalist as a scholarship athlete on the USC womens rowing team After graduating from USC, Ms. Zeanah chose to puruse a Master of Science in applied physiology and kinesiology with a concentration in exercise physiology at the University of Florida and to continue research in skeletal muscle physiology. For the next three years, Ms. Zeanah conducted in vitro skeletal muscle research in the Molecular Physiology Laboratory directed by Dr. David Criswell. During her third year, she also began doctoral program studies in exercise physiology and conducted clinical and t ranslational cardiovascular research in the Cardiovascular Physiology Laboratory under the direction of Dr. Randy Braith. After obtaining her Master of Science degree from the University of Florida in 2009, Ms. Zeanah plans to continue her studies and car diovascular research in pursuit o f a Doctor of Philosophy degree.