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Glucocorticoid-Induced Myopathy is Mediated by Impaired Nitric Oxide Synthesis

Permanent Link: http://ufdc.ufl.edu/UFE0019672/00001

Material Information

Title: Glucocorticoid-Induced Myopathy is Mediated by Impaired Nitric Oxide Synthesis
Physical Description: 1 online resource (67 p.)
Language: english
Creator: Long, Jodi Heather D
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

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

Notes

Abstract: Glucocorticoid drugs are potent and widely prescribed anti-inflammatory agents. However, glucocorticoid-induced skeletal muscle wasting severely limits the efficacy of these drugs, especially in chronic treatment situations. Understanding the mechanisms behind this muscle-wasting side effect will lead to more effective countermeasures, thus improving quality of life in various patient populations. The systemic effects of glucocorticoids are mediated, in part, by inhibition of inducible nitric oxide synthase (iNOS). Nitric oxide (NO) production by muscle serves important signaling functions. Therefore, NOS down-regulation in skeletal muscle may contribute to glucocorticoid-related myopathy. Based on this, we hypothesized glucocorticoid use inhibits NO production in skeletal muscle, and that this inhibition is partly responsible for the atrophy noted with glucocorticoid use. Furthermore, we hypothesized that addition of L-Arginine (L-Arg), a NOS substrate, and DETA-NONO, an NO donor would attenuate the glucocorticoid effects. We chronically (8wk) treated mice with prednisolone or saline at a concentration of 2.1mg/kg of body weight. At the end of the treatment period the animals were euthanised and their gastrocnemius removed for either Western Blot analysis or Single Fiber Isolation and culture. We demonstrated chronic exposure of mice to glucocorticoids decrease muscle satellite cell activity and NOS expression. In vitro treatment with the nitric oxide donor, DETA-NONO, restored satellite cell activity in myofibers isolated from glucocorticoid-treated mice to control levels. Additionally, we cultured L6 myotubes to study the acute effects of glucocorticoids. After 24h of treatment (100?M dexamethasone), the myotubes displayed 6-fold increased expression of MAFbx, a muscle specific ubiquitin ligase. Co-treatment with DETANONO or L-Arg blunted the effect. Following 48h of treatment myotubes dimensions were assessed. The glucocorticoid treatment reduced myotube area by over 50%. When DETANONO or L-Arg was added, myotube atrophy was significantly attenuated. Thus, NO may be a useful therapeutic target for opposing the negative effects of both acute and chronic glucocorticoid use.
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 Jodi Heather D Long.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Criswell, David S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0019672:00001

Permanent Link: http://ufdc.ufl.edu/UFE0019672/00001

Material Information

Title: Glucocorticoid-Induced Myopathy is Mediated by Impaired Nitric Oxide Synthesis
Physical Description: 1 online resource (67 p.)
Language: english
Creator: Long, Jodi Heather D
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

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

Notes

Abstract: Glucocorticoid drugs are potent and widely prescribed anti-inflammatory agents. However, glucocorticoid-induced skeletal muscle wasting severely limits the efficacy of these drugs, especially in chronic treatment situations. Understanding the mechanisms behind this muscle-wasting side effect will lead to more effective countermeasures, thus improving quality of life in various patient populations. The systemic effects of glucocorticoids are mediated, in part, by inhibition of inducible nitric oxide synthase (iNOS). Nitric oxide (NO) production by muscle serves important signaling functions. Therefore, NOS down-regulation in skeletal muscle may contribute to glucocorticoid-related myopathy. Based on this, we hypothesized glucocorticoid use inhibits NO production in skeletal muscle, and that this inhibition is partly responsible for the atrophy noted with glucocorticoid use. Furthermore, we hypothesized that addition of L-Arginine (L-Arg), a NOS substrate, and DETA-NONO, an NO donor would attenuate the glucocorticoid effects. We chronically (8wk) treated mice with prednisolone or saline at a concentration of 2.1mg/kg of body weight. At the end of the treatment period the animals were euthanised and their gastrocnemius removed for either Western Blot analysis or Single Fiber Isolation and culture. We demonstrated chronic exposure of mice to glucocorticoids decrease muscle satellite cell activity and NOS expression. In vitro treatment with the nitric oxide donor, DETA-NONO, restored satellite cell activity in myofibers isolated from glucocorticoid-treated mice to control levels. Additionally, we cultured L6 myotubes to study the acute effects of glucocorticoids. After 24h of treatment (100?M dexamethasone), the myotubes displayed 6-fold increased expression of MAFbx, a muscle specific ubiquitin ligase. Co-treatment with DETANONO or L-Arg blunted the effect. Following 48h of treatment myotubes dimensions were assessed. The glucocorticoid treatment reduced myotube area by over 50%. When DETANONO or L-Arg was added, myotube atrophy was significantly attenuated. Thus, NO may be a useful therapeutic target for opposing the negative effects of both acute and chronic glucocorticoid use.
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 Jodi Heather D Long.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Criswell, David S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0019672:00001


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8042149fbe5519b47882848a5685cb5c6b4525df







GLUCOCORTICOID-INDUCED MYOPATHY IS MEDIATED BY IMPAIRED NITRIC
OXIDE SYNTHESIS




















By

JODI HEATHER DIXON LONG


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

UNIVERSITY OF FLORIDA

2007

































O 2007 Jodi Heather Dixon Long









ACKNOWLEDGMENTS

I thank the chair and members of my supervisory committee for their mentoring and the

members of the Molecular Physiology Lab for their support and technical assistance. I thank Dr.

Randy Braith and Kathy Howe for their collaborative efforts in this proj ect. Also, I thank my

family for their support and understanding as I completed my study.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S ................. ...............3.......... ......


LI ST OF T ABLE S ................. ...............6................


LIST OF FIGURES .............. ...............7.....


AB S TRAC T ......_ ................. ............_........9


CHAPTER


1 INTRODUCTION ................. ...............11.......... ......


Questions to be Addressed............... ...............1
Hypotheses............... ...............1
h r Yivo .............. ...............13~~~~
hr Yitro.......... ...............14~~~~~~~

Significance .............. ...............14....

2 REVIEW OF LITERATURE ................. ...............15....___ ....


Glucocorticoids and Muscle Remodeling. ................. ............ ........__. ........ 1
Proteolytic Systems Involved in Skeletal Muscle Remodeling ................. .........__ ........16
IGF-1 Pathway and Muscle Remodeling ................. ...............19...............
Satellite Cells and Skeletal Muscle Remodeling ................. ...............21...............
Nitric Oxide and Skeletal Muscle Remodeling .............. ...............22....
Glucocorticoids and Nitric Oxide............... ...............24.
Concluding Statement ................. ...............25........ ......


3 MATERIALS AND METHODS .............. ...............26....


hr Yitro Studies ........._ ........_. ...............26...

Experimental Design .............. ...............26....
Cell Culture .............. ...............26....
Hi stochemi stry ........._._ .......__. ...............27...
Nitric Oxide Production .............. ...............28....
W western Blots .............. .......... ............ .... .......2
RNA Isolation and Quantitative Real-Time PCR .............. ...............29....
hIn [ vo Studies ........._._ .......__. ...............30...

Experimental Design ....................... ...............3
Protein Isolation and Western Blotting .............. ...............31....
mRNA Measurements .............. ...............32....

Single Fiber Isolation .............. ...............32....
Statistical Analysis .............. ...............34....












4 RE SULT S .............. ...............36....


In Vitro Studies .............. .. ..._ ...............36...
MAFbx mRNA Expression .............. ...............36....
CAT and NOS mRNA Expression ................. ...............36........... ...
NOS and a-Spectrin Protein Content .............. ...............36....
Histological Measurements .............. ...............36....
Nitric Oxide Production .............. ...............37....
In Vivo Studies................ ..... .. .... ..... .... .........3

Body and Muscle Masses and Total Protein Content ......____ ........._ ................37
MAFbx, NOS, and CAT mRNA Expression .............. ...............37....
NO S Protein Content ................. ...............3.. 8......... ....
Satellite Cell Emanation ................. ...............38........... ....


5 DI SCUS SSION ................. ...............45................


Acute Effects of Glucocorticoids ........ ................. ...............45 ....

Myotube Dimensions............... ...............4
NO Availability .............. ...............46....
Calpain Activation............... ...............4
M AFbx Control .................... .... ....... ..............4
Possible Mechanisms of Action for Nitric Oxide. ................ .............. ........ .....48
CAT Expression in Cultured Myotubes .............. ...............49....
Chronic Effects of Glucocorticoids .............. ...............50...
NOS Expression in Mouse Skeletal Muscle............... ...............50.
CAT Expression in Mouse Skeletal Muscle............... ...............51.
Satellite Cell Activity in Single Muscle Fibers ................. ...._.._ .............. .....5


6 CONCLU SION................ ..............5


LI ST OF REFERENCE S ....._.._................. ........_.._.........5


BIOGRAPHICAL SKETCH .............. ...............66....










LIST OF TABLES


Table page

3-1 Cell diagram illustrating experimental design of the cell culture portion of proposed
proj ect. ............. ...............3 5....

3-2 Cell diagram illustrating experimental design of the isolated myofiber portion of the
proposed proj ect ................. ...............35........... ....

4-1 mRNA data for MAFbx in control and dexamethasone treated cells............... ................39

4-2 mRNA data for CAT transporters in cell culture ................. ...............39........... .

4-3 Body and muscle masses and total protein content of animals ................. ............... ....39

4-4 mRNA data for CAT transporters in animals .............. ...............39....










LIST OF FIGURES


Figure page

4-1 Protein analysis ot-spectrin. ............. ...............40.....

4-2 Representative images from cells fixed in 3.9% formaldehyde and H & E stained..........41

4-3 Average area of individual myotubes. ................ ........._.__........ ...._.__.......42

4-4 Nuclear number per myotube............... ...............42

4-5 Protein analysis nNOS and eNOS. ............. ...............43.....

4-6 Satellite cells emanating from activated fibers. ............. ...............44.....

5-1 Illustration of the muscle maintenance pathways ................ ...............53........... ..

5-2 Illustration of potential effects of nitric oxide. ................ ................. ..............53









LIST OF ABBREVIATIONS

CAT: Cationic amino acid transporter

MAFbx: Muscle Atrophy F-box

NO: Nitric oxide

eNOS: Endothelial nitric oxide synthase

iNOS: Inducible nitric oxide synthase

nNOS: Neuronal nitric oxide synthase









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

GLUCOCORTICOID-INDUCED MYOPATHY IS MEDIATED BY IMPAIRED NITRIC
OXIDE SYNTHESIS

By

Jodi Heather Dixon Long

August 2007

Chair: David S. Criswell
Maj or: Health and Human Performance

Glucocorticoid drugs are potent and widely prescribed anti-inflammatory agents.

However, glucocorticoid-induced skeletal muscle wasting severely limits the efficacy of these

drugs, especially in chronic treatment situations. Understanding the mechanisms behind this

muscle-wasting side effect will lead to more effective countermeasures, thus improving quality

of life in various patient populations.

The systemic effects of glucocorticoids are mediated, in part, by inhibition of inducible

nitric oxide synthase (iNOS). Nitric oxide (NO) production by muscle serves important

signaling functions. Therefore, NOS down-regulation in skeletal muscle may contribute to

glucocorticoid-related myopathy. Based on this, we hypothesized glucocorticoid use inhibits NO

production in skeletal muscle, and that this inhibition is partly responsible for the atrophy noted

with glucocorticoid use. Furthermore, we hypothesized that addition of L-Arginine (L-Arg), a

NOS substrate, and DETA-NONO, an NO donor would attenuate the glucocorticoid effects.

We chronically (8wk) treated mice with prednisolone or saline at a concentration of

2.1Img/kg of body weight. At the end of the treatment period the animals were euthanised and

their gastrocnemius removed for either Western Blot analysis or Single Fiber Isolation and

culture. We demonstrated chronic exposure of mice to glucocorticoids decrease muscle satellite









cell activity and NOS expression. In vitro treatment with the nitric oxide donor, DETA-NONO,

restored satellite cell activity in myofibers isolated from glucocorticoid-treated mice to control

levels. Additionally, we cultured L6 myotubes to study the acute effects of glucocorticoids.

After 24h of treatment (100C1M dexamethasone), the myotubes displayed 6-fold increased

expression of MAFbx, a muscle specific ubiquitin ligase. Co-treatment with DETANONO or L-

Arg blunted the effect. Following 48h of treatment myotubes dimensions were assessed. The

glucocorticoid treatment reduced myotube area by over 50%. When DETANONO or L-Arg was

added, myotube atrophy was significantly attenuated. Thus, NO may be a useful therapeutic

target for opposing the negative effects of both acute and chronic glucocorticoid use.









CHAPTER 1
INTRODUCTION

Muscle mass is determined by a delicate balance between protein synthesis and

breakdown, which is keenly sensitive to active muscle tension and the pattern of muscle

recruitment. Skeletal muscle atrophy, denoted by a decrease in muscle mass and fiber size, can

arise from various etiologies including denervation, disuse, sepsis, cancer, AIDS, and chronic

exposure to high levels of glucocorticoids. The latter could arise from adrenal hyperactivity

(Cushing's Disease) or pharmacological administration of glucocorticoids as anti-inflammatory

agents. Regardless of the initiating factor, atrophy is characterized by a decrease in protein

synthesis and an increase in protein degradation, with the latter having a larger impact. And,

rather than simply an absence of growth or maintenance signals, muscle wasting is an active

remodeling mechanism by which skeletal muscle myofibrils are dismantled and removed.

Understanding this mechanism will lead to more effective strategies to attenuate muscle atrophy,

thus improving quality of life in various patient populations.

Glucocorticoid administration is linked to skeletal muscle atrophy (33, 34, 75) and

increased expression of genes involved in proteolysis (17, 19, 37, 105). Additionally, the

inhibition of calcium-activated proteases (calpains) in glucocorticoid treated myotubes attenuates

the usual glucocorticoid-induced protein loss and decrease in myotube size (23). Therefore,

glucocorticoids have the capacity to affect multiple components of the atrophy program.

Three maj or proteolytic systems have been identified in skeletal muscle: the lysosomal,

calpain, and ubiquitin-proteasome pathways. The lysosomal system is unlikely to be involved

because it does not degrade myofibrillar proteins (24, 54, 111). It has been shown in multiple

catabolic situations that contributions to atrophy via the lysosome are minimal (91, 99, 102).









However, calpain and ubiquitin-proteasomal pathways seem to be intimately involved with the

protein degradation associated with atrophy.

The ubiquitin-proteasomal pathway is primarily responsible for degrading myofibrillar

proteins into component amino acids. However, it does not degrade intact myofibrillar proteins

(46, 84). Thus, myofibrillar loss initially is dependent on disassembly of myofibrils, which can

occur via calpain activity. The dismantled myofibrils are then targeted to the proteasome for

degradation, resulting in protein loss. Glucocorticoids activate both the calpain and the

proteasome pathways (22, 23, 76, 100).

Nitric oxide, a soluble gas product of L-arginine metabolism via nitric oxide synthase

(NOS) activity, has rather global impacts on skeletal muscle. It is necessary for muscle

differentiation (53), myoblast fusion (50, 67), satellite cell activation (2, 3, 93, 94), growth (78),

and maintenance of muscle mass (44, 106). In fact, calpain activity, which initiates protein

degradation, is inhibited by basal NO production (44). Attenuation of endogenous nitric oxide

production in skeletal muscle may be an important mechanism of glucocorticoid-induced

myopathy since NO production and expression of NOS enzymes are known to be downregulated

in the presence of glucocorticoids (7, 41, 43). In preliminary studies, we have found a dramatic

deficit in satellite cell number and responsiveness to mechanical activation in muscle fibers

isolated from glucocorticoid-treated mice. Furthermore, 24h of treatment with DETA-NONO (an

NO donor) can attenuate this satellite cell deficit (Betters et al., unpublished data). Therefore,

the loss of NO production in skeletal muscle may initiate proteolysis via calpain and/or

proteasome activation. Concomitantly, loss of NO may compromise the ability of skeletal muscle

to maintain muscle mass in the long-term via a loss of satellite cell number and/or activity.









Cellular availability of nitric oxide or its biochemical precursors may offer a convenient and

effective target for treatment and prevention of glucocorticoid-induced myopathy.

Therefore, this proj ect will employ two experimental models, daily inj section of

prednisolone for 8 wks in adult mice and 24 or 48h treatment of cultured C2C12 mouse or L6 rat

myotubes with dexamethasone, to examine the effects of glucocorticoid treatment on skeletal

muscle NOS expression, calpain activity, expression of the muscle-specific ubiquitin E3 ligase,

MAFbx, and abundance and activity of satellite cells. Further, treatment of cultured adult

myofibers and L6 myotubes with the NO donor, DETA-NONO, and the NOS substrate, L-

arginine will test the postulate that glucocorticoid-induced myopathies are related to

compromised NO production. The potential for NO supplementation to ameliorate the negative

effects of glucocorticoids on skeletal muscle will be assessed.

Questions to be Addressed

1. What is the effect of glucocorticoid treatment on NOS protein expression and cationic amino
acid transporter (CAT) mRNA expression in mouse skeletal muscle?

2. What is the effect of glucocorticoid treatment on NO production (nitrate accumulation), NOS
protein expression, and CAT and MAFbx mRNA expression in myotubes?

3. What is the effect of glucocorticoid treatment on calpain activation?

4. What is the effect of glucocorticoid treatment on satellite cell activation?

5. Can nitric oxide supplementation in glucocorticoid treated myotubes inhibit MAFbx
expression, calpain activity, and atrophy?

6. Can L-arginine supplementation ameliorate glucocorticoid-induced MAFbx expression,
calpain activity, and atrophy in myotubes, due to enhanced nitric oxide production?

Hypotheses

Inz Vivo

1. Daily inj sections of prednisolone for 8 wks will decrease NOS protein and CAT mRNA
expression in the gastrocnemius muscle of mice.










2. Prednisolone treatment (8 wks) will reduce the number and activation/proliferation of
satellite cells in single myofibers isolated from the mouse gastrocnemius muscle.

3. Treatment of isolated myofibers from prednisolone-treated mice with the NO donor, DETA-
NONO, will increase the activation/proliferation of satellite cells in response to a mechanical
stimulus.

In2 Vitro

4. NO or L-arginine supplementation in dexamethasone-treated myotubes will increase NOS
protein and CAT mRNA expression, limit MAFbx expression, and inhibit atrophy.

5. NO or L-arginine supplementation in dexamethasone-treated myotubes will decrease the
abundance of calpain-specific proteolytic fragments of co-spectrin.

Significance

The debilitating consequences of glucocorticoid-induced myopathies are of clinical

relevance. Although the pathological overproduction of cortisol (i.e. Cushing's Disease) is

relatively rare, synthetic glucocorticoids are widely used pharmacologically for their dramatic

anti-inflammatory effects. The loss of skeletal muscle can lead to loss of independence, falls or,

potentially, respiratory failure. Thus, it is critical we understand the mechanisms that cause

atrophy in response to chronic glucocorticoid treatment so that better countermeasures can be

designed. Clearly nitric oxide is involved in normal skeletal muscle development, function, and

hypertrophy (2, 67, 78, 83), but its role in attenuating atrophy is unclear. The attenuation of

endogenous nitric oxide production due to glucocorticoid-induced loss of nNOS protein may

represent an important signal initiating the atrophy process. If so, supplementation with the NOS

substrate, L-arginine or with a pharmacological nitric oxide donor could be an effective strategy

to ameliorate glucocorticoid-induced skeletal muscle myopathy. This study is designed to

examine this possibility and contribute to our understanding of the mechanisms involved in

muscle wasting.









CHAPTER 2
REVIEW OF LITERATURE

Skeletal muscle is a highly adaptive tissue. It is sensitive to not only changes in use, but

also to various common pharmacological treatments. Muscle mass is determined by a delicate

balance between protein synthesis and breakdown, and is capable of responding to increased or

decreased loading by increasing (hypertrophy) or decreasing (atrophy) mass accordingly.

Unfortunately, certain disease states (e.g. cancer, sepsis) and/or pharmacological agents (e.g.

glucocorticoids) can induce rapid and severe muscle wasting. This skeletal muscle atrophy,

denoted by a decrease in muscle mass and fiber size, is characterized by a decrease in protein

synthesis and an increase in protein degradation, with the latter having a more prominent role. It

is not wholly clear how glucocorticoids exert their effects, but their administration has been

linked to increased expression of genes involved in proteolysis (17, 19, 37, 105). Therefore,

delineating the mechanism of glucocorticoid-induced atrophy is key to developing

countermeasures to this potentially debilitating condition.

Glucocorticoids and Muscle Remodeling

Glucocorticoids are a group of steroid hormones that have receptors throughout the body,

thus their effects impact a number of physiological systems. The most widely understood effects

are on carbohydrate metabolism and immune function. This class of hormones stimulates

gluconeogenesis in the liver and increases amino acid mobilization from non-hepatic tissues.

Additionally, they inhibit glucose uptake in muscle, and stimulate lipolysis in adipose tissue.

Clearly these hormones elicit a catabolic effect in skeletal muscle. Despite the deleterious

effects of chronic exposure to glucocorticoids, they are widely used pharmacologically for their

anti-inflammatory and immunosuppressant qualities.









Glucocorticoids have been shown to cause proteolysis in several situations (59).

Treatment of C2C12 cells with dexamethasone, a synthetic glucocorticoid, not only inhibits

proliferation (96), but also induces atrophy in terminally differentiated myotubes (7, 75, 89). In

addition to protein degradation, dexamethasone administration induces MAFbx and MuRF 1

expression (59, 75, 105). The up-regulation of these transcription factors was blocked by a

pharmacologic inhibitor of glucocorticoids (113). Furthermore, glucocorticoid-induced MAFbx

and MuRF1 expression seems to be operating through FOXO activation (76, 87), which is

directly related to the PI3K/Akt pathway.

In addition to the increase in components of the ubiquitin-proteasome pathway,

dexamethasone increases intracellular concentrations of calcium in myocytes (Evenson et al.

2005, unpublished observation). This could in turn lead to increased calpain activity and may be

one of many ways glucocorticoids exert their wasting effects on skeletal muscle. Another

potential path is via inhibition of IGF-1 signaling. In the absence of IGF-1, Akt is

dephosphorylated which leads to FOXO activation (76), thus promoting atrophy. Additionally, it

should be noted that diminished Akt signaling promotes the cleavage and activation of caspase-3

(85). IGF-1 has been shown to attenuate the effects of glucocorticoids on L6 cells (52). In fact,

Latres et al. (48) demonstrated an inverse regulation of MAFbx dependent on IGF-1 presence

and determined that both the Akt/FOXO and Akt/mTOR pathways are needed for transcriptional

changes induced by IGF-1.

Proteolytic Systems Involved in Skeletal Muscle Remodeling

Three maj or proteolytic systems have been identified in skeletal muscle: the lysosomal,

calpain, and ubiquitin-proteasome pathways. The lysosomal system is unlikely to be involved in

atrophy because it does not degrade myofibrillar proteins (24, 54, 111). It has been shown in

multiple catabolic situations that contributions to atrophy via the lysosome are minimal (91, 100,










102). However, calpain and ubiquitin-proteasomal pathways seem to be intimately involved

with the protein degradation associated with atrophy.

The calpains (calcium-activated proteases) are comprised of a family having at least 14

members. Some of these are ubiquitous enzymes, such as CL- and m-calpain, while others are

tissue specific proteins, such as the muscle specific calpain 3, also called p94 (28, 86, 90). The

C1- and m-calpain isoforms are named based on the calcium concentration needed to attain their

half-maximal proteolytic activation. The C1-calpain form requires CIM concentrations and the m-

calpain requires mM concentrations (5, 16, 20). Both levels of calcium are supraphysiological

(16, 55), so some mechanism exists to activate the normally inactive calpains. This is evident in

that calpains are relevant in physiological systems (55), as demonstrated by the work showing

that in vivo calpain inhibition has a protective effect on skeletal muscle (70, 101).

The calpains are activated in skeletal muscle during reduced use (79, 91). When activated

these proteases target structural proteins within the muscle cell such as talin, a linker protein in

the cell membrane (44). The dismantling of the cytoskeleton via calpain activation seems to be a

critical event in cellular protein degradation (27, 39).

The regulation of calpain activity is complex. The most important activator of calpain is

calcium (28). Cytoplasmic calcium is increased in glucocorticoid treated cell cultures (8, 48,

103). Furthermore, Wei et al. (109) has shown that calpain inhibitor (calpeptin) administration

blocks dexamethasone-induced proteolysis. Thus, one potential role dexamethasone plays in the

atrophic response is to increase cytosolic calcium with subsequent calpain activation.

Calcium is not the only regulator of calpain-mediated proteolysis. Calpastatin (an

endogenous inhibitor of calpains) and nitric oxide may play a regulatory role (44). Calpastatin

prevents calpain enzymatic activation and the expression of catalytic activity (27). Nitric oxide









has been shown to regulate sarcoplasmic reticulum function and therefore intracellular calcium.

This action inhibited calpain action in ischemia-reperfused rat hearts (14). Zhang, Kraus, and

Truskey (1 14) showed NO inhibited calpain-mediated proteolysis of talin in C2C12 myotubes.

Thus, calpains are regulated by a number of factors.

Calpain activity is likely not the sole contributor to atrophy in the presence of elevated

glucocorticoids. The role of the ubiquitin-proteasome pathway in atrophy cannot be discounted.

The cellular events involved in this method of protein breakdown are highly coordinated. First,

the protein to be degraded is marked by attaching ubiquitin to it. This is accomplished via a

trifecta of proteins: El, the ubiquitin-activating enzyme, E2, the ubiquitin-conj ugating enzyme,

and E3, the ubiquitin-protein ligases (e.g., MAFbx or atrogin-1 and MURFl1) (40). El activates

ubiquitin and transfers it to E2. Then, E3 transfers the ubiquitin from E2 to the target protein.

This occurs multiple times until the protein to be degraded is marked by a chain of ubiquitin

molecules. Once polyubiquinated, the targeted protein is rapidly degraded by the proteasome

(26, 58, 98).

Atrophy is partly a result of the ubiquitin-proteasome pathway. In both the skeletal muscle

of immobilized rat hindlimb and atrophied cardiac muscle, mRNA for MAFbx and MuRF 1 were

increased (47, 71). Ikemoto et al. (40) demonstrated an increase in ubiquinated proteins within

skeletal muscle during reduced use. There is a concomitant increase in proteasome enzyme

activity (79). It has been demonstrated by a number of authors that this system accounts for most

of the elevated muscle proteolysis in a variety of atrophic situations (36, 91, 95) including

glucocorticoid use (38, 112).









IGF-1 Pathway and Muscle Remodeling

IGF-1 (insulin-like growth factor-1) is a growth factor that initiates a cascade of

intracellular signaling events leading to hypertrophic and antiproteolytic responses. Two main

pathways stimulated by IGF-1 are the Ras-Raf-1VEK-ERK pathway and the PI3K/Akt pathway.

The Ras-Raf-1VEK-ERK signaling events affect fiber type composition, but not myofiber size

(62), thus this pathway is likely unimportant in causing hypertrophy or atrophy. However, the

PI3K/Akt pathway seems to cause muscle hypertrophy ultimately by stimulating translational

events via the mTOR and GSK kinases (12).

mTOR, one target of Akt, when phosphorylated will activate p70S6 kinase. This kinase

increases muscle protein translation, ultimately supporting hypertrophy. Recently Thompson

and Gordon (97) demonstrated that older rats subj ected to skeletal muscle overload had less

phosphorylation of mTOR and its downstream target p70S6K that correlated with diminished

total protein content. Further evidence of a hypertrophic role of mTOR was shown by rapamycin

blocking myotube hypertrophy in a C2C12 model of terminally differentiated myotubes (63, 65).

The Park et al. group further demonstrated the necessity of mTOR for hypertrophy by stably

expressing in C2C12s rapamycin-resistant forms of mTOR and p70S6K. The rapamycin-

resistant cultures underwent hypertrophy with rapamycin treatment, while the control cultures

did not hypertrophy. This was assessed by image analysis. An additional role mTOR plays in

muscle remodeling is through inhibition of FOXO (forkhead box O), which targets at least one of

the E3 ligases. Based on the evidence presented here, mTOR, when phosphorylated by Akt will

cause hypertrophy and inhibit atrophy (25, 49).

Akt also directly targets the forkhead box O (FOXO) family of transcription factors, which

target at least one of the E3 ligases. Phosphorylated Akt inactivates (phosphorylates) FOXO,

thus hindering protein degradation. FOXO, when active, translocates to the nucleus and









increases transcription of a key element of the ubiquitin-proteasome pathway, the ubiquitin

ligase atrogin-1 or MAFbx. The functional importance of this protein being expressed during the

atrophy process was demonstrated by using MAFbx-/- mice. Mice lacking the MAFbx gene had

less atrophy following denervation (11). This was the same for the other E3, MuRF1 in a

MuRFl1-/- knockout mouse model. Furthermore, because MuRF 1 and MAFbx have been shown

to be upregulated in other models of atrophy, they are widely accepted as reliable markers of the

atrophy process (11, 12, 29, 51, 113). So, under the influence of IGF-1 through Akt signaling,

the activity of FOXO is repressed while that of mTOR is increased (12). This causes an increase

in cell size through a concomitant increase in protein synthesis and a decrease in protein

degradation. Alternatively, in the absence of IGF-1, there is an increase in proteolysis and the

expression of atrophy-related ubiquitin ligases such as MAFbx, correlated with FOXO

dephosphorylation (activation) (76).

FOXO factors are critical in the transcription of MAFbx in myotubes under both starvation

and glucocorticoid treated conditions (76). In both situations, IGF-1 signaling decreases. Thus

phosphorylation of FOXO via Akt activation is minimized (13, 64, 92), and FOXO translocates

to the nucleus (9, 13) to up-regulate MAFbx expression (76), and hence induce atrophy. Further

support for the necessity of nuclear FOXO in atrophic conditions was established when a

constitutively active form of FOXO3 (one of the three mammalian FOXOs associated with

skeletal muscle) remained in the nucleus and caused atrophy (76, 82, 88).

In addition to Akt regulating protein balance in skeletal muscle via FOXO and mTOR, it

also targets glycogen synthase kinase (GSK3P). GSK3P is blocked by phosphorylated (active)

Akt, and leads to hypertrophy (72). However, when GSK3P is activated, it blocks transcriptional

initiation factors necessary for protein synthesis (12). This would indicate GSK3 P is a potential










mitigator of skeletal muscle atrophy. However, Sandri et al. (76) demonstrated that activation of

GSK3 P does not influence the induction of MAFbx. So, perhaps GSK3 P's role in muscle

metabolism is limited to its control of initiation factors involved in protein synthesis.

The maj ority of the evidence regarding atrophy in skeletal muscle indicates that down-

regulation of IGF-1 signaling is a key event leading to proteolysis. There seem to be two parts to

the atrophic response via this mechanism. First, definitive links have been established regarding

nuclear localization of FOXO factors in the absence of IGF-1 signaling and also FOXO factors

binding directly to the MAFbx promoter to cause an increase in this ubiquitin ligase (76).

Second, Song et al. (85) showed that down-regulation of IGF-1 signaling via the Akt/mTOR

pathway leads to calpain activation. It is fairly widely recognized that calpains likely dismantle

the existing cytoskeleton, readying targeted myoproteins for ubiquination and subsequent

degradation via the proteosome.

Satellite Cells and Skeletal Muscle Remodeling

Adult skeletal muscle fibers consist of post-mitotic, terminally differentiated nuclei. Each

of these nuclei govern a Einite volume of cytoplasm, the size of which seems to be set and tightly

regulated (i.e. the nuclear domain hypothesis) (31). In order for skeletal muscle fibers to recover

from injuries or adapt their size to meet functional demands, and still maintain this nuclear

domain range, a source of nascent myonuclei must be available. Muscle satellite cells, which are

quiescent muscle precursor cells found between the myofibers and the external lamina, provide

this source. In response to growth or regeneration stimuli, these cells are activated to

differentiate and join an existing myofiber or form a new myotube, thus maintaining the

myonuclear domain.









Activation of satellite cells is a necessary step in adult skeletal muscle growth. Several

studies have used localized gamma irradiation of rat skeletal muscles to eliminate mitotically

active cells within the muscle, without compromising circulating stem cells or growth factors

(66, 73). These irradiated muscles are completely incapable of regaining muscle mass following

injury (60, 73) or growing in response to mechanical overload (1, 73). This suggests that addition

of nascent myonuclei is required for muscle growth rather than simply following changes in fiber

size.

In addition to supporting muscle growth and regeneration, satellite cells may be important

for maintenance of muscle mass. Atrophied muscles from hindlimb suspended animals contain

fewer satellite cells associated with isolated myofibers (61). It seems that repeated use of

myogenic precursor cells for muscle repair can lead to exhaustion of the satellite cell pool (18,

108). Additionally, it appears that these same satellite cells from atrophied animals have an

impaired ability to activate and proliferate (61). As myonuclei are removed during atrophy, the

down-regulation of satellite cell number and activity likely prevents the addition of new nuclei to

maintain the fiber size. Perhaps this de-activation of satellite cells is a primary mechanism to

inhibit hypertrophy and induce atrophy.

Nitric Oxide and Skeletal Muscle Remodeling

Nitric Oxide (NO), a product of L-arginine (L-Arg) metabolism, is a free radical that is

produced by nitric oxide synthase (NOS) enzymes. All three isoforms of the NOS enzyme can be

expressed in skeletal muscle. nNOS is most abundant, being associated with the dystrophin

complex and localized in costameres at the sarcolemma (30). eNOS is also constitutively

expressed in muscle and may be associated with mitochondria (42). Lastly, iNOS is believed to

be only expressed in response to an inflammatory stimulus (87). Although NO is primarily

known for its vasodilatory effects, it is also an important regulatory molecule in many different









tissues, including skeletal muscle (87). Our lab has demonstrated that nitric oxide (NO)

positively influences skeletal muscle hypertrophy and contractile gene expression during

overload (78, 83). Anderson (2) found that NO is a primary signal for skeletal muscle satellite

cell activation. Others have shown nitric oxide to be important in myoblast fusion (50, 67).

Thus, it is clear that NO is involved with the hypertrophic response.

NO production increases in isolated rat glomeruli when incubated with IGF-1 (107).

Additionally, an increase in Akt phosphorylation was noted. This rise in Akt phosphorylation

associated with NO presence has also been demonstrated in bovine aortic endothelial cells (56).

Furthermore, when an mTOR inhibitor was used in activated macrophages, NO production was

reduced (110). While these studies are not in skeletal muscle, they demonstrate a potential link

between NO production and the PI3K/Akt/mTOR signaling pathway.

In addition to nitric oxide's influence on or production from hypertrophic pathways, it has

also been associated with components of the atrophic pathway. For example, Koh and Tidball

(44) showed evidence that NO could be a regulatory molecule of calpains. They used sodium

nitroprusside, an NO donor, and showed no proteolysis of structural proteins. Moreover, through

zymography and an activity assay, inhibition of m-calpain in C2C12 cultures was found.

Michetti et al. (57) also showed inhibition of m-calpain in a dose-dependent manner with sodium

nitroprusside. Further support for a role of NO in calpain inhibition comes from Chohan et al.

(14). They demonstrated that ischemia-reperfused hearts had an increase in calpain activity and

a decrease in cytosolic NO levels. When L-Arg was administered, the increased calpain activity

was attenuated. Thus, whether NO was directly given or synthesized via one of the NOS

enzymes and L-Arg, it inhibits calpain activity, suggesting that some basal endogenous NO

production may inhibit proteolysis and support maintenance of muscle mass.










Endogenous NO production is dependent upon L-Arg uptake (80). L-Arg is transported

into the cells via a sodium-independent y+ transport system. This system recognizes cationic

amino acids, specifically L-Arg, L-Lys, and ornithine (15). There are three known transporters

for L-Arg present in skeletal muscle: CAT-1, CAT-2A, CAT-2B. CAT-1 and CAT-2B have a

high affinity for L-Arg compared to CAT-2A. There is some evidence that NO production in

skeletal muscle may be limited by L-Arg availability. Since plasma levels of L-Arg generally

exceed the Km value for the NOS enzymes, an L-Arg limitation would implicate the CAT

transporters as potential regulators of NO production.

Glucocorticoids and Nitric Oxide

Glucocorticoids, known for their atrophy-inducing qualities, suppress NO production (41,

80). In fact, much of the anti-inflammatory actions of glucocorticoids are likely due to inhibition

of iNOS. Dexamethasone inhibits iNOS transcription and mRNA expression (6, 45). Further,

glucocorticoids have been shown to induce proteolytic cleavage of iNOS at a specific site within

the calmodulin binding domain via activation of calpain (103). This domain is conserved among

the three NOS isoforms, raising the interesting possibility that glucocorticoids may globally

down-regulate NO production, simultaneously compromising the signaling actions of nNOS

and/or eNOS while inhibiting the inflammatory effects of iNOS. In cardiac microvascular

endothelial cells, treatment with glucocorticoids completely abolishes NO production, an effect

attributed to eNOS down-regulation (80). eNOS has been shown to be down-regulated by

FOXO1 and FOXO3A (68). These factors bind to the eNOS promoter in endothelial cells. This

causes repression of eNOS expression.

Glucocorticoids may also affect NO production via L-Arg availability. Simmons et al.

(80) showed a reduction in L-Arg uptake in dexamethasone treated cells. Dexamethasone

treatment prevented the induction of CAT-2B mRNA by cytokines. Furthermore, Hammermann









et al. (32) found a down-regulation of CAT-2B in rat alveolar macrophages after 20h exposure to

dexamethasone. It seems that the inflammatory response increases L-Arg transport into the cells,

which supports large-scale NO production by iNOS. Dexamethasone, however, is able to not

only attenuate this increase in L-Arg influx by the down-regulation of CAT transporters, but also

seems to suppress iNOS (81). Therefore, glucocorticoids have the ability to greatly reduce NO

production by inhibiting both substrate availability and NOS enzyme expression. Given the

importance of NO signaling for skeletal muscle remodeling as discussed above, inhibition of

endogenous NO production may be an important mechanism for glucocorticoid-induced skeletal

muscle myopathy.

Concluding Statement

Our lab has demonstrated that nitric oxide (NO) positively influences skeletal muscle

hypertrophy. Koh and Tidball (44) found that NO inhibition of calpains protected cells from

proteolysis. Wang et al. (106) inhibited NO production via L-NAME administration and induced

atrophy. Moreover, glucocorticoid use can decrease NO production, increase intracellular

concentrations of calcium and induce expression of MAFbx. Taken together, it is not

unreasonable to believe that glucocorticoid-induced skeletal muscle myopathy is due, at least in

part, to compromised NO production. An NO-donor and/or supplementation with the NOS

substrate L-Arg could attenuate glucocorticoid-induced atrophy. This study explored the effects

of glucocorticoid treatment on NOS and CAT expression in skeletal muscle using both in vivo

and in vitro models. Further, the potential for an NO donor or L-Arg supplementation to inhibit

muscle atrophy via down-regulation of MAFbx expression and calpain activity, and improved

satellite cell function was experimentally tested.









CHAPTER 3
MATERIALS AND METHODS

The purpose of this proj ect was to examine the mechanism of glucocorticoid-induced

skeletal muscle myopathy, and test the hypothesis that the loss of satellite cell activity and

initiation of the calpain and proteasome proteolytic pathways during exposure to glucocorticoids

is mediated by the loss of endogenous nitric oxide production.

Inz Vitro Studies

Experimental Design

The C2C12 mouse and L6 rat myogenic cell lines were used to examine the effects of

exposure to the glucocorticoid, dexamethasone. Differentiated myotube cultures were treated

with dexamethasone for 48h and monitored for myotube dimensions, protein content, and

proteolytic cleavage of the calpain substrate, ot-spectrin. Alternatively cultures were treated for

24h and assessed for expression of the ubiquitin ligase MAFbx (or atrogin-1) and nitric oxide

production. Supplementation of cultures with DETA-NONO, a nitric oxide donor, or L-arginine,

the NOS substrate, were performed to test for a causal relationship between compromised nitric

oxide production and initiation of the atrophy program. Table 3-1 illustrates the design of this

study .

Cell Culture

C2C12 and L6 cells (ATCC, Manassas, VA) were plated and proliferated in Dulbecco' s

Modified Eagle's Medium (DMEM) growth media (GM) containing 10% FBS and 1%

penicillin/ streptomycin. At 80% confluency the GM was removed and the cells washed twice

with 370C PBS. Then differentiation media (DM) was added (DMEM supplemented with 2%

horse serum and 1% penicillin/ streptomycin). Cultures differentiated for 5-7 days until there

were confluent myotubes. At that point cultures were divided into 3 groups and treated for 24h










or 48h with one of the following: 10 mM L-arginine, 10CIM DETA-NONO, or no supplement

(control) in DM. Half of the cultures from each group were co-treated with 100CLM

dexamethasone.

At the time of harvest, cultures were rinsed twice with ice cold PBS and then harvested in

two manners. For protein analysis via Western Blots, the cells treated for 48h were harvested on

ice in 0.300ml non-denaturing lysis buffer (NDL: 20% Triton X-100; IM Tris/ pH=7.5; 5M

NaCl; 0.5M EDTA; 10mg/ml sodium azide; 4mg/ml NaF; 4ug/ml NaVO3; 0.1% protease

inhibitors) and then centrifuged at 500 x g for 5min to remove insoluble material. The

supematant was used for Western Blots for nNOS, eNOS, and a-spectrin. For mRNA analysis,

parallel cultures treated for 24h were harvested in 1ml of ice cold TRIzol (Invitrogen, Carlsbad,

CA).

Histochemistry

Parallel cultures were Eixed with 3.7% formaldehyde and stained with hematoxylin and

eosin at 48h. Microphotometric digital images of each culture were captured using a Zeiss

Axiovert200 light microscope (Thomwood, NY) and Qimaging RETIGA EXi digital camera

(Surry, BC, Canada) and software (IPLab3.6.5, Scanalytics, Rockville, MD). The images were

evaluated for myotube dimensions (area and nuclei/ area) and density (myotube number/ Hield of

view) using Scion Image imaging software. Three images of each culture were captured.

Within each image 10 myotubes were analyzed for area and nuclear content by measuring the

first 10 myotubes in each Hield of view beginning in the left-hand comer. Thus, 30 myotubes

were analyzed per culture and each treatment had three cultures for a total n = 90 myotubes per

treatment.









Nitric Oxide Production

Nitric oxide production by the cells was measured by assessing nitrate accumulation in

media measured at Oh, 24h, and 48h. Following collection, the media was centrifuged at 5000 x

g for 40min using microcentrifuge filters to remove serum, which can interfere with the kit

components. The Nitrate/Nitrite Fluorometric Assay Kit from Cayman Chemical Company

(Ann Arbor, MI) based on the procedures described by Misko et al. (1993) was used.

Western Blots

Myotube protein was collected as described above. Protein concentration was determined

by DC assay (Bio-Rad, Rockville Centre, NY). SDS-PAGE was performed on 7%

polyacrylamide gels. Equal amounts of protein were loaded into each lane. Positive controls for

nNOS and eNOS were used. The gels were run at 60V for 1h and then 100V for 1h (NOS blots)

or 100V for 2h (u-spectrin blots) with a 1h transfer at 500mA. The transfer was onto

nitrocellulose membranes that were subsequently blocked in Odyssey blocking buffer for 1h.

The membranes were then incubated overnight with primary antibody in 1:1 TB S-T (0.01%):

Odyssey blocking buffer. For nNOS and eNOS, a 1:500 dilution was used (monoclonal, mouse,

anti-nNOS: 611852, eNOS: 610328, BD Transduction Laboratories, San Jose, CA). For a-

spectrin (Biomol), a 1:5000 dilution was used. p-actin incubation was used as a loading control

(1:4000 dilution). Following the overnight incubation at 4oC, the membranes were washed 4

times for 5min each with TBS-T. The fluorescent secondary antibody (anti-mouse HRP,

Amersham, Piscataway, NJ) was applied to the a-spectrin membrane using a 1:3000 dilution,

while the nNOS blot was incubated with anti-rabbit using a 1:8000 dilution. All blots were then

incubated at room temperature for 35min. Then membranes were washed four times for 5min

each with TB S-T and two additional washes with TB S. The membranes were scanned on the










Odyssey infrared imaging system (Li-Cor, Lincoln, Nebraska) and the relative fluorescence of

the bands was quantified by densitometry using the accompanying software. Results are

expressed as mean + SEM.

RNA Isolation and Quantitative Real-Time PCR

We examined expression of mRNA transcripts for iNOS, the two cationic amino acid

transporters responsible for L-Arg uptake in skeletal muscle cells (CAT-1 and CAT-2), and

MAFbx.

Total RNA was extracted from cultured cells by harvesting in 1 ml of TRIzol (Invitrogen,

Carlsbad, CA) according to the manufacturer' s instructions. Concentration and purity of the

extracted RNA was measured spectrophotometrically at A260 and A280 in lX TE buffer

(Promega, Madison, WI). Purified RNA was then stored at -800 C for later assay.

Reverse transcription (RT) was performed using the Super Script III First-Strand Synthesis

System for reverse transcription-polymerase chain reaction (RT-PCR) according to the

manufacturer's instructions (Life Technologies, Carlsbad, CA). Reactions were carried out using

1 Cpg of total RNA and 2.5 CLM oligo(dT)20 primerS. First strand cDNA was treated with two

units of RNase H and stored at -800 C.

Primers and probes for CAT-1, CAT-2, iNOS, and MAFbx (assay # Mm00432019_ml,

Mm00432032_ml, Mm00440485_ml, and Rn00591730, respectively) were obtained from the

ABI Assays-on-Demand service and consisted of Taqman 5' labeled FAM reporters and 3'

nonfluorescent quenchers. Primer and probe sequences from this service are proprietary and

therefore, are not reported. Primer and probe sequences also consisting of Taqman 5' labeled

FAM reporters and 3' nonfluorescent quenchers for hypoxanthine guanine phosphoribosyl

transferase (HPRT) obtained from Applied Biosystems (Assays-by-Design) are: Forward, 5'-









GTTGGATACAGGCCAGACTTTGT-3'; Reverse, 5'-AGTCAAGGGCATATCCAACAACAA

-3'; Probe, 5'-ACTTGTCTGGAATTTCA-3'.

Quantitative real-time PCR was performed using the ABI Prism 7700 Sequence Detection

System (ABI, Foster City, CA). Each 25 Cl1 PCR reaction, performed in duplicate, contained 1 Cll

of cDNA reaction mixture. In this technique, amplification of the fluorescently labeled probe

sequence located between the PCR primers was monitored in real-time during the PCR program.

The number of PCR cycles required to reach a pre-determined threshold of fluorescence (called

the CT) was determined for each sample. Samples were quantified relative to the CT for a

normalizing gene determined separately in the same sample. This procedure is referred to as the

comparative CT method as described by Bustin (2002). HPRT was selected as the appropriate

normalizer since the expression of this gene in C2C12 cells is not significantly altered during

differentiation and fusion (p > 0.05).

In2 Vivo Studies

Experimental Design

Adult mice were treated with the glucocorticoid, prednisolone, for 8 wks to examine the

effects on skeletal muscle mass, protein content, satellite cell abundance and activity, and the

potential for a nitric oxide donor to rescue prednisolone-induced deficits in satellite cell

activation/proliferation in isolated, cultured skeletal muscle fibers.

Animals. Seven-month old male Swiss-Webster mice were obtained from Harlan (N=16).

After a seven-day acclimation period, mice were weighed and then given a subcutaneous

inj section daily, six days per week in the morning for eight weeks. They received either Depo-

Medrol (prednisolone; Pharmacia & Upj ohn, Kalamazo, MI; n=8) or sterile saline (n=8) at a

concentration of 2.1Img/kg of body weight. All inj sections were prepared so that each inj section

was approximately a 0.1Iml volume. Food (normal mouse chow given ad libitum) was weighed









every three days for the duration of the study to account for weight loss due to reduced food

consumption. Animals were weighed weekly on the non-injection day, and treatment dosage

was adjusted for the next week based on the current animal weight. They were housed one to a

cage in SPF-2 at ACS, University of Florida, under normal conditions (12h light/dark cycle). On

the last day of the study they were anesthetized with inhaled isoflurane (2-3.5%) with oxygen as

the carrier gas using an anesthesia cart (with the charcoal filter scavenger attached). Euthanasia

was by exsanguination and was confirmed with cervical dislocation. Following this, the

animal's skin and superficial fascia of the hind limbs was removed to expose the muscles of the

hind limbs. The gastrocnemius (GN) muscle was dissected out and weighed immediately. In

four animals per group, the muscle was flash frozen in liquid nitrogen for subsequent analysis of

muscle protein content and Western blot procedures. The gastrocnemius from the remaining

four animals from each group was digested with collagenase for myofiber isolation. (IACUC

protocol #E401)

Protein Isolation and Western Blotting

Frozen muscles were thawed and homogenized in buffer (0.1M Tris Base; 0. 1M NaPO4,

0.01M EDTA; 30% glycerol; pH=7.8) with protease inhibitors (200mM Benzamidine; Img/ml

Pepstatin A and Aprotinin; 5mg/ml Leupeptin; IM DTT; 40mM phenylmethyl sulfonyl) at a 1:5

dilution based on muscle weight. This was done on ice, with the dismemberator on for two bouts

of 10sec each. Following homogenization, the slurry was allowed to sit on ice for 10min. Then

100ul was removed and added to 700ul of 0.05M NaOH and left overnight at room temperature

for total protein versus connective tissue assay. The remainder of the slurry was centrifuged at

750 x g for 15min at 40C. The supernatant was transferred to clean microcentrifuge tubes and

stored at -800C until used for Western Blotting.









Total protein and connective tissue protein was measured in the homogenized samples.

Following an overnight digest (at least 18h) in 0.05M NaOH, total protein was assessed using the

DC assay (Bio-Rad) and then the samples were centrifuged at 4000 x g for 15min to pellet the

connective tissue and then the supernatant was subjected to the protein assay again. The second

assessment of protein content is considered to be the protein in the non-connective tissue

fraction. This measurement was used to determine equal protein loading for the Westemn Blots.

Western blot analysis for nNOS and eNOS in chronically treated muscle was done by

methods described previously but the protein was transferred to a PVDF membrane. These

membranes were blocked in 5% non-fat dry milk in TBS-T (0.01%) for 1h. They were rinsed in

TBS-T and incubated overnight at a 1:500 dilution polyclonall anti-nNOS, Cayman Chemical,

Ann Arbor, MI; polyclonal anti-eNOS BD Transduction Laboratories, San Jose, CA). Secondary

antibodies (anti-rabbit and anti-mouse for nNOS and eNOS, respectively; Sigma, St. Louis, MO)

were applied for 1h (1:5000). Protein bands were detected with ECL+ (Amersham Biosciences,

UK). The relative amounts of the bands were quantified by densiometry using ImageJ software.

mRNA Measurements

Total RNA was isolated from gastrocnemius (GN) samples after homogenization in Trizol.

Reverse transcription followed by quantification of iNOS, CAT-1, and CAT-2 mRNA by

quantitative real-time PCR was performed as described above for the cell culture experiment.

Single Fiber Isolation

GN muscles (N = 4 animals/group) were surgically removed by first removing the skin of

the hindlimbs, exposing the Achilles tendon. Then a small incision was made behind the knee

and forceps and small sharp scissors were used to cut away the upper layer of fascia and

connective tissue down the length of the muscle. As much as possible of the fascia was carefully

peeled away using a scalpel. Then the tendons at the knee were cut on either side at an angle.









Without pulling on the muscle, the Achilles tendon was cut and the GN pulled up while carefully

trimming connective tissue on either side. This was done on both limbs. Once the GNs were

removed, they were rinsed in PBS, filleted, and then immediately transferred to a 15ml falcon

tube containing digest media (9ml DMEM, 1% penicillin/ streptomycin, Iml collagenase). The

digest was rocked at 370C for 1.5h. Following the digest, the muscles were titurated three times

with a wide-bore 10ml disposable pipet, then the fibers were allowed to settle. After that, the

digest media was removed with a pipet down to about the 3ml mark. Then about 10ml of fresh,

pre-warmed media (DMEM, 10% FBS, 1% penicillin/ streptomycin) was added to the falcon

tube containing the fibers, and the tube inverted a few times. The media was removed again and

the rinse repeated 1-2 more times. This diluted the collagenase. At this point 3-4ml of the digest

was transferred to a series of pl00 plates containing warm media. Quality fibers were removed

from the surrounding dead (hypercontracted) fibers and debris by transferring to a clean plate

(containing 15ml media) using a wide-bore 200C1l pipet. Plates were not out of the incubator

longer than 10min during this process. Once the chosen fibers were ready to plate, Iml of warm

media was added to each well of a 24-well plate and the plate was allowed to equilibrate in the

incubator for 10-15min. Then a drop of matrigel was applied to each well. The fiber was added

by extracting them from the clean plates in 50C1l volume using a p200 pipet tip (with the end

snipped-off) and another drop of matrigel was applied. After all fibers were plated, the 24-well

plate was placed in the incubator for at least 15min. Then the plate was centrifuged at 1100 x g

at 370C for 30-40min to activate the satellite cells. The fibers were maintained at 370C, 5% CO2,

for 24h. Treatments of DETA-NONO (0, 5, 10, 50C1M) were added to the fibers from both

control and glucocorticoid treated animals following the centrifugation step. After 24h, the

cultures were fixed in 2% formaldehyde and immunohistochemistry performed for MyoD and









DAPI staining. The number of myogenic cells was quantified to assess activation or

proliferation of satellite cells. Table 3-2 illustrates this design.

Statistical Analysis

Statistical analyses were performed on SPSS (version 12.0.1) using 2-way ANOVAs

(glucocorticoid vs. supplement). Post hoc analyses were performed using Tukey's test.

Significance was established at p<0.05.










Table 3-1. Cell diagram illustrating experimental design of the cell culture portion of proposed
proj ect.
Dexamethasone
(100 CIM)


No Supplement Control
L-Arginine (10 mM)
DETA-NONO (10 C1M)


n= 6
n= 6
n= 6


n= 6
n= 6
n= 6


Table 3-2. Cell diagram illustrating experimental design of the isolated myofiber portion of the
proposed proj ect.


Fibers isolated from
Control mice
(n=4 mice)
~100 fibers

~100 fibers
~100 fibers
~100 fibers


Fibers isolated from
Prednisolone-treated mice
(n=4 mice)
~100 fibers

~100 fibers
~100 fibers
~100 fibers


No Supplement
Control
5 C1M DETA-NONO
10 CIM DETA-NONO
50 CIM DETA-NONO









CHAPTER 4
RESULTS

Inz Vitro Studies

MAFbx mRNA Expression

Dexamethasone treatment of L6 myotubes resulted in a six-fold increase in MAFbx

mRNA expression compared to control (Table 4-1). Supplementation of arginine (10mM) or

DETA-NONO (5pLM) significantly blunted the effect of the dexamethasone, even though

MAFbx mRNA did not reach control levels (Table 4-1). Treatment of the L6 myotubes with

arginine alone significantly reduced MAFbx expression, whereas DETA-NONO

supplementation alone showed a trend toward lower MAFbx expression but did not reach

statistical significance (Table 4-1). The interaction between treatment groups was significant at

all levels (p<0.05). Thus, the upregulation of MAFbx seen with dexamethasone use may be

attenuated by L-Arg or DETA-NONO.

CAT and NOS mRNA Expression

In C2C12 cells, the mRNA expression for both transporters (CAT-1 and CAT-2) was

significantly depressed in glucocorticoid treated cells (p<0.05) (Table 4-2). CT values for iNOS

were below detectable limits.

NOS and a-Spectrin Protein Content

There was no significant difference in the cleaved/total a-spectrin protein expression

normalized to p-actin with any treatment in cell culture (p=0.818) (Figure 4-1). nNOS and

eNOS expression in both L6s and C2C12s was not detectable.

Histological Measurements

Size, nuclear number, and total myotube area per field of view were decreased in

glucocorticoid-treated cells (Figure 4-2). The total myotube area per field of view dropped by









26% after exposure to dexamethasone, and this effect was attenuated with arginine treatment

(only an 8.1% decrease when L-Arg was added). The dexamethasone effect was more moderate

when DETA-NONO was added (23% decrease). Not only was less of the field of view covered

by myotubes in the glucocorticoid-treated cells, these myotubes were also smaller (64%). When

DETA-NONO or L-Arg was added to the dexamethasone-treated cells, the myotubes were only

42% smaller than controls (Figure 4-3). Nuclear number was significantly decreased by

dexamethasone treatment (p<0.05), and this was attenuated when L-Arg or DETA-NONO was

added to the medium (Figure 4-4). While confirming dexamethasone causes atrophy, these data

also indicate that NO may partially attenuate the loss in size and nuclear number.

Nitric Oxide Production

Nitric oxide production was measured in vitro via 24h nitrate accumulation in the media.

Media was assessed at time Oh, 24h, and 48h. Regardless of the treatment or time point, there

were no differences between groups for fluorescence. So, cells treated with dexamethasone may

not have compromised NOS activity.

Inz Vivo Studies

Body and Muscle Masses and Total Protein Content

Body mass and gastrocnemius mass did not significantly decline with 8 weeks of

prednisolone treatment (Table 4-3). Total protein/gastrocnemius muscle did not significantly

decrease, but there was a trend to be lower in glucocorticoid muscle compared to control.

MAFbx, NOS, and CAT mRNA Expression

MAFbx mRNA levels are expected to have reached a steady state of expression after 8

weeks of glucocorticoid treatment, thus this was not analyzed in muscle homogenate. However,

CAT and iNOS expression were considered. CAT expression was insignificantly elevated in the










glucocorticoid treated animals (Table 4-4). iNOS values were below detectable limits, and thus

not reported.

NOS Protein Content

The expression of nNOS and eNOS protein was reduced by over 30% in the glucocorticoid

treated animals compared to controls (Figure 4-5B). Representative blots are displayed in Figure

4-5A.

Satellite Cell Emanation

MyoD+ satellite cells emanating from isolated myofibers 48 hours after centrifugation

were significantly reduced with in vivo prednisolone treatment (54% of control fibers had

emanating cells present vs. 25% of glucocorticoid fibers, Figure 4-6). However, supplementation

of culture media with DETA-NONO (5-50 CIM) induced satellite cell emanation in a dose-

dependent manner. Fifty CIM DETA-NONO eliminated the difference in centrifuge-induced

satellite cell emanation between glucocorticoid and control fibers (76% of control vs. 74% of

glucocorticoid). This suggests that NO bioavailability limits satellite cell activity in response to a

mechanical stress. Further, exogenous NO can rescue compromised satellite cell function

following glucocorticoid treatment.









Table 4-1. mRNA data for MAFbx in control and dexamethasone treated cells. Values are
means f SE. Transcripts are normalized to hypoxanthine guanine phosphoribosyl
transferase (HPRT) mRNA and expressed relative to control value. Significantly
different from *control and "fdexamethasone (within group); p<0.05.
Treatment Dex (-) Dex (+)
Control 1.040f0.149 6. 122f0.408*
L-Arg (10mM) 0.807f0.000* 4.321f0.426*?
DETA-NONO (10CIM) 0.716f0.088* 3.904f0.476*?

Table 4-2. mRNA data for CAT transporters in cell culture. Values are means f SE. Transcripts
are normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA
and expressed relative to control value. Significantly different from *control; p<0.05.
Gene of interest Control Glucocorticoid
CAT-1 2.534 f 1.229 0.107 f 0.071*
CAT-2 1.503 f 0.3945 0.242 f 0.129*

Table 4-3. Body and muscle masses and total protein content of animals. Values are means f
SE. There was no value significantly different from control; body mass p=0.170,
gastrocnemius mass p=0.490, total protein/ muscle p=0.087.
Control Glucocorticoid
Body Mass (g) 36.4 & 0.95 34.4 & 1.18
Gastrocnemius Mass (g) 150.2 & 5.75 154.8 & 3.96
Total Protein (mg)/ Muscle (g) 15.81 & 0.74 13.61 & 0.90

Table 4-4. mRNA data for CAT transporters in animals. Values are means f SE. Transcripts are
normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA and
expressed relative to control value. No value was significantly different from control
(CAT-1 p=0.221; CAT-2 p=0.18).
Gene of interest Control Glucocorticoid
CAT-1 1.740 f 0.699 3.146 f 0.820
CAT-2 1.115 f 0.281 2.002 f 0.548













t240kDa

150~kDa


CI *rr --


.... C


A) 46kDa





g0.004-
a0.003-





C Dex

B) treatments



Figure 4-1. Protein analysis a-spectrin A) representative immunoblot for a-spectrin (total
240kDa; cleaved 150kDa) and p-actin (loading control; 46kDa) in L6 myotubes
treated with 24h dexamethasone or vehicle. Lanes 1 4 are control, lanes 5 8 are
Dexamethasone (Dex). B) quantification of cleaved-to-total a-spectrin ratio. No
value was statistically significant (p=0.818).















Fiur 4-.Rpeettv mgsfrmclsfxdi .%frmleyeadH&Esand
A) onro B dxamthsoe De) ) 1mML-rgD)Dex+ 0m LAr E
10M EA-OO ) e +1pMDEANOO















7000
6000
5000
4000
3000
2000
1000
0


gControl
g Dex


Arg NO
Treatments


Figure 4-3. Average area of individual myotubes. Quantification of myotube area. Values are
means & SE. Significantly different from *control and "fdexamethasone; p<0.05.





30


S25

S20

15

10


gControl
Dex


C Arg
Treatments


Figure 4-4. Nuclear number per myotube. Quantification of nuclear number. Values are means
SSE. Significantly different from *control and "fdexamethasone; p<0.05.

















IILi~k


nNO S


1 200


1 200

1 000


1 000

S0 800

S0 600

S0 400

0 200


,0800

S0 600

0 400

0 200


0 000


0 000


CON


CON


Group


Group


nNO S


eNOS


Figure 4-5. Protein analysis nNOS and eNOS A) representative immunoblot for nNOS and
eNOS in mouse muscle homogenate treated with 8wk prednisolone or vehicle. B)

quantification of nNOS and eNOS normalized to control.


eNOS













'5 + 60.00-

r'~ C Control
t o 40.00-
om ~oGC
2 2i 30.00-



10.00

0 CtM 5 CtM 10 CtM 50 CtM
DETA-NO Concentration

Figure 4-6. Satellite cells emanating from activated fibers. Quantification of cells emanating
from activated fibers. Supplementation of culture media with DETA-NONO (5-50
CIM) induced satellite cell emanation in a dose-dependent manner.









CHAPTER 5
DISCUSSION

Glucocorticoids cause substantial skeletal muscle atrophy (34, 77) and suppress NO

production (42, 81). We show that 24h of treatment with glucocorticoids in cell culture increases

MAFbx mRNA expression and that an NO donor attenuates the increased expression. After 48h

of glucocorticoid treatment, we witness a dramatic decrease in myotube dimensions, which is

partially rescued by either L-Arg or nitric oxide donation. We failed to confirm our hypothesis

that NO production is compromised in glucocorticoid treated cells, as there was no change in

nitrate accumulation in 24h media collection.

In addition to these acute effects observed with glucocorticoid use, we demonstrate that

eight weeks of corticosteroid treatment reduces satellite cell activation/ proliferation from

isolated fibers, which is necessary for skeletal muscle remodeling. Moreover, nNOS and eNOS

protein content are reduced in glucocorticoid treated gastrocnemius. Interestingly, we found NO

donation restores satellite cell emanation from glucocorticoid treated fibers to levels of control

fibers, suggesting NO availability may be partially responsible for glucocorticoid-induced

myopathy, in vivo.

Acute Effects of Glucocorticoids

Myotube Dimensions

Glucocorticoids induce protein loss and a decrease in myotube size (23). We show that

48h dexamethasone treatment reduces myotube dimensions a decrease in size of 64%.

Additionally, dexamethasone treatment resulted in less coverage of the field of view by

myotubes and in fewer nuclei per myotube. We hypothesized that the atrophic myotubes were a

result of reduced NO availability.









Initially to test this, we supplemented cultures with either L-Arg (10mM) or DETA-NONO

(10CIM). With these additions, the reduction in myotube size associated with dexamethasone

treatment was attenuated by 22%. Furthermore, supplement inclusion resulted in maintenance of

nuclear number. These results suggest, at least in part, that glucocorticoid-induced atrophy can

be rescued with nitric oxide.

NO Availability

While the histological morphology data imply that NO availability may be compromised,

these measurements fail to specifically demonstrate a reduction in NO production by

glucocorticoid treated cells. In order to test this hypothesis we indirectly measured NO

production by assessing nitrate and nitrite accumulation in the media. Fluorescence was constant

in all samples. As we were unable to measure changes in nitric oxide production, we cannot

confirm a limitation in the skeletal muscle's ability to produce nitric oxide. And, even though

our attempt to verify NOS protein in L6 lysate was inconclusive, we are confident these cells

possess this protein (10), and thus are capable of synthesizing nitric oxide.

Calpain Activation

Glucocorticoid administration likely induces skeletal muscle atrophy through increased

expression of genes involved in proteolysis (17, 19, 38, 106). The inhibition of calcium-

activated proteases (calpains) in glucocorticoid treated myotubes attenuates the usual

glucocorticoid-induced protein loss and decrease in myotube size (77). And while it is generally

accepted that calpain activation is a key initial event in protein degradation, we failed to show

this in atrophy associated with glucocorticoid use. We showed atrophy in glucocorticoid treated

myotubes versus controls can occur without a concomitant increased in cleaved versus total ot-

spectrin, a structural protein targeted by calpains. This is in agreement with Banik et al. (4), who









found a methylprednisolone-induced dose-dependent inhibition of rabbit muscle calpain activity.

They also demonstrated enzyme inhibition with dexamethasone and prednisolone treatments.

Another group (25) found a decrease in calpain activity and a-spectrin cleavage in mechanically

ventilated rat diaphragm following glucocorticoid treatment. Additionally, Wang et al. (106)

demonstrated only a small contribution of calcium-dependent muscle proteolysis in L6 myotube

dexamethasone-induced atrophy. Furthermore, groups showing a positive correlation between

dexamethasone treatment and calpain activity were actually measuring enzymatic activity. We

analyzed the result of calpain activity in the living cell by measuring cleaved versus total a-

spectrin protein, as the measurement of in vitro maximal enzymatic activity may not represent in

vivo proteolytic activity. Further measurements will be necessary to draw firm conclusions

regarding the role of calcium-dependent muscle proteolysis in glucocorticoid-induced myopathy.

MAFbx Control

MAFbx has at least three controls. It is regulated by two elements of the IGF-1/ Akt

pathway and by one component of the atrophy pathway (Figure 5-1). Akt has two routes for

diminishing MAFbx expression. It blocks FOXO, which is directly responsible for upregulation

of MAFbx and enhances mTOR activity, which not only increases p70S6K, but also blocks

MAFbx (26, 50). In addition to these events in the hypertrophy pathway that inhibit MAFbx

expression, it has been shown that p38 via TNFu signaling in the atrophy pathway stimulates

MAFbx expression.

We have demonstrated that 24h of treatment with glucocorticoids in cell culture increases

MAFbx mRNA expression and that an NO donor attenuates the increased expression. It is not

totally clear why an upregulation of MAFbx is seen with glucocorticoid use. IGF-1 and insulin

both stimulate the hypertrophy pathway (Figure 5-1), and insulin resistance develops with









glucocorticoid use (70). Also it has been shown that NOS is essential for skeletal muscle

hypertrophy (84). So, perhaps instead of solely increasing activity of the atrophy pathway, the

hypertrophic signals are attenuated, thus removing the blockade on FOXO and MAFbx via

deactivated Akt, with the ultimate effect of dramatic skeletal atrophy.

Nitric oxide's attenuation of the glucocorticoid effect on MAFbx mRNA is likely though

the hypertrophy pathway, as it has been shown that NOS is essential for skeletal muscle

hypertrophy (84).

Possible Mechanisms of Action for Nitric Oxide

Nitric oxide has been shown to be a key regulator of muscle hypertrophy and satellite cell

activation. It has not been completely elucidated how nitric oxide is having its effect, but

perhaps it is through some component of the hypertrophy pathway. This would explain its

ability to attenuate glucocorticoid-induced atrophy. We have shown that adequate NO

availability may be a limitation in skeletal muscle myopathy following glucocorticoid treatment.

The fact that we observed dramatic recovery of myotube dimensions and nuclear number

and partial attenuation of MAFbx mRNA leads us to believe NO is potentially acting on a

downstream target of the hypertrophy (IGF-1) pathway. For example, in arteries with functional

endothelial cells, IGF-1 caused a concentration-dependent relaxation (78). This effect was

abolished by the use of a NOS inhibitor. Futhermore, this author demonstrated formation of NO

in response to IGF-1 treatment in cultured endothelial cells, and that the signaling was

independent of intracellular Ca2+, but involved PI3K, a direct downstream target of IGF-1. PI3K

is required to phosphorylate (activate) Akt. Perhaps glucocorticoids attenuate Akt

phosphorylation, which would lower activation of mTOR and activate FOXO thereby leading to

atrophic signaling through MAFbx. Supplementation with exogenous L-arginine or nitric oxide

reverses the atrophy by suppressing MAFbx activation.









Furthermore, MAFbx may be directly upregulated with glucocorticoid use due to FOXO

activation. GSK3 P is blocked by phosphorylated (active) Akt, and leads to hypertrophy (73) and

FOXO inactivation (Figure 5-2). Sandri et al. (77) has shown that this same pathway, when

inhibited at the IGF-1 level, will increase FOXO activation. We show the upregulation of

MAFbx seen with glucocorticoid treatment was moderately attenuated by use of L-arginine, a

NOS substrate, and NO donation (via DETA-NONO). Thus, the mild attenuation of MAFbx

upregulation seen with the treatment of NO or L-Arg could be a result of any one of multiple

elements in the IGF-1 pathway. Due to the lack of apparent calpain activation, which seems to

not involve PI3K, it would be more reasonable to think that NO is working at least at the Akt

level, if not further downstream. Further studies are necessary to determine if Akt and or FOXO

are directly involved in NO action in skeletal muscle maintenance.

CAT Expression in Cultured Myotubes

The reduced expression of the CAT transporters seen in cell culture agrees with Simmons

et al. (81) who showed a reduction in L-Arg uptake in dexamethasone treated cells.

Dexamethasone treatment prevented the induction of CAT-2B mRNA by cytokines. In further

agreement with our findings, Hammermann et al. (33) found a down-regulation of CAT-2B in rat

alveolar macrophages after 20h exposure to dexamethasone. It seems that the inflammatory

response increases L-Arg transport into the cells, which supports large-scale NO production by

iNOS. Dexamethasone, however, is able to not only attenuate this increase in L-Arg influx by

the down-regulation of CAT transporters, but due to its anti-inflammatory characteristics also

seems to suppress iNOS (82). Our evidence supports this in that CAT-2 mRNA expression was

down-regulated with dexamethasone treatment, however we were unable to measure changes in

NOS. Therefore, glucocorticoids have the ability to reduce NO production by inhibiting









substrate availability through lower arginine transport into the cell. Given the importance of NO

signaling for skeletal muscle remodeling as discussed above, inhibition of endogenous NO

production may be an important mechanism for glucocorticoid-induced skeletal muscle

myopathy. Futhermore, if the mechanism to synthesize NO is compromised with glucocorticoid

treatment, then an NO donor should attenuate the effects of the glucocorticoid better than

substrates for NO synthesis. This phenomenon may explain how DETA-NONO was a superior

to L-Arg for inhibiting MAFbx expression. L-Arg only decreased the glucocorticoid-induced

MAFbx mRNA expression by 1.8 fold, while DETA-NONO had a marginally better effect of a

2.3 fold decrease in expression. Without as much NOS or CAT present, physiological levels of

L-Arg might not have been sufficient. But, bypassing the NOS enzyme, as was done using the

NO donor DETA-NONO, a greater effect was observed.

Chronic Effects of Glucocorticoids

NOS Expression in Mouse Skeletal Muscle

After 8 weeks of prednisilone administration, we report a drop in both eNOS and nNOS

expression in mouse skeletal muscle. This suggests that corticosteroids actually lead to lower

amounts of the enzyme, which could result in reduced nitric oxide production and possible

changes in both hypertrophic and atrophic signaling pathways.

eNOS down-regulation seems to be the cause of the lack of NO production observed with

glucocorticoid treatment in cardiac microvascular endothelial cells (81). eNOS has been shown

to be down-regulated by FOXO1 and FOXO3A (69). These factors bind to the eNOS promoter

in endothelial cells. This causes repression of eNOS expression, and subsequent NO generation.

So, the acute effects of glucocorticoids may be upregulation of MAFbx expression via FOXO

action, with FOXO being the culprit of the long-term effect of decreased NOS expression.









CAT Expression in Mouse Skeletal Muscle

NOS presence is not the sole factor in determining NO production. L-Arg, a substrate for

NOS, must be transported into the cell via the CAT transporters. These transporters had greater

mRNA expression in the animals. Perhaps the increase in expression seen in the animals was

due to a more chronic exposure to the corticosteroid. As NOS expression continued to decline

over the 8 weeks, an upregulation in the cationic transporter was possibly an attempt to counter

the decrease in eNOS and nNOS and restore NO levels. Since we did not directly measure NO

production, we cannot confirm the net effect of decreased NOS expression and increased CAT

transporter expression. Nevertheless, the efficacy of NO supplementation to isolated myofibers

implies that endogenous NO production may have been compromised in the GC group.

Satellite Cell Activity in Single Muscle Fibers

In addition to NO inhibiting the synthesis of MAFbx and thus diminishing the initial

atrophy effect, it can also be used to rescue satellite cells from glucocorticoid treated animals. It

has been shown by numerous labs that activation of satellite cells is a necessary step in adult

skeletal muscle growth and regeneration (1, 61, 67, 74). Futhermore, our work confirms these

cells may be critical for maintenance of muscle mass. Other labs have demonstrated this in

atrophied muscles from hindlimb suspended animals. Isolated myofibers from these animals

contained fewer satellite cells and these cells had an impaired ability to activate and proliferate

(62). This de-activation may be yet another factor in atrophy. In our study, we found a

significant reduction in satellite cells emanating from glucocorticoid-treated fibers in response to

mechanical stimulation. Approximately half of the fibers from control animals had at least one

emanating myoD+ mononuclear cell, while only one-fourth of the fibers from glucocorticoid

treated animals had emanating satellite cells.









Not only did we confirm that satellite cell function is compromised with chronic

glucocorticoid treatment, but we also show exogenous NO treatment can ameliorate the

glucocorticoid-induced deficit in satellite cell activation/ proliferation. In other models this has

been a successful treatment for satellite cell activation (2, 3, 94, 95). Our findings suggest that

NO production may be reduced with prolonged glucocorticoid exposure, and this is what reduces

satellite cell activity.









SAtrophy Pathway


Inflammatory
Cvtokines



38


IGF-1


insulin








Cleaves actin


t NF -B

T C1/2 FOXO


eIF2B $1MAFbx MUJRF1

p70S6K


Figure 5-1. Illustration of the muscle maintenance pathways




L-Arg


Figure 5-2. Illustration of potential effects of nitric oxide.


SHypertrophy Pathway I









CHAPTER 6
CONCLUSION

Glucocorticoid treatment both in vitro and in vivo has detrimental effects. Acutely, we

confirmed they cause upregulation of a key element of the Ub-prot pathway (MAFbx). Chronic

treatment decreases constitutive NOS, thus potentially compromising NO production. NO has

been shown to be expressed in conjunction with activation of the IGF-1 pathway and critical to

satellite cell activity, both of which play a role in muscle mass maintenance. We demonstrate

that NO attenuates the expression of MAFbx with acute glucocorticoid treatment. Furthermore

we show that NO can rescue satellite cell activity from muscle chronically treated with

glucocorticoids. So, NO may impact both the atrophic and hypertrophic pathways in its role in

muscle mass maintenance. NO supplementation to cultures did have positive effects on

maintaining myotube dimensions and increasing satellite cell activity. So, even if it does not

directly inhibit a component of the atrophy pathway, NO may be a viable treatment option by

augmenting muscle mass maintenance from the hypertrophy side via satellite cell activity and

fusion with the existing myotubes. Thus, enhancing NO production may be important in

attenuating the atrophic effects associated with both acute and chronic glucocorticoid use.









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BIOGRAPHICAL SKETCH

Jodi Heather Dixon Long was born in DuPage County, Illinois. The older of two children

she grew up mostly in Carol Stream, Illinois, but moved with her family to St. Simons Island,

Georgia in 1985 where she graduated salutatorian of the class of 1989 from Frederica Academy.

In 1993, she earned her B.A. in mathematics, with a minor in secondary education from Erskine

College in Due West, S.C. Upon baccalaureate graduation, Jodi served as a middle and high

school math and science teacher and taught group exercise.

In 1997, Jodi returned to school to earn a M. S. in exercise physiology from Georgia

Southern University. She was a graduate assistant in the physical education program while

earning this degree. Following graduation in December of 1998, Jodi returned to a teaching

career. She taught physical activity classes and anatomy and physiology at Coastal Georgia

Community College in Brunswick, GA. In the fall of 1999, Jodi moved to a full time instructor

position at her alma mater of Georgia Southern University where she taught anatomy and

physiology, exercise physiology and health survey courses.

Once again, Jodi returned to school to complete her terminal degree. In the fall of 2002,

she began studies at the University of Florida under the direction of David S. Criswell. While

studying at UF, Jodi also taught anatomy and physiology at Santa Fe Community College in

Gainesville, FL, eventually becoming a full time faculty member.

Following graduation from the doctor of philosophy program in the College of Health and

Human Performance (Department of Applied Physiology and Kinesiology), Jodi plans to

continue to educate college students in anatomy and physiology at Santa Fe Community College.

She also continues to teach group fitness.









In addition to her professional and academic achievements, Jodi was married in 1991 to

George W. "Trey" Long, III of McCormick, SC and they have two children, Sam (6yr) and Alex

(4yr) .





PAGE 1

GLUCOCORTICOID-INDUCED MYOPATHY IS MEDIATED BY IMPAIRED NITRIC OXIDE SYNTHESIS By JODI HEATHER DIXON LONG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1

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2007 Jodi Heather Dixon Long 2

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ACKNOWLEDGMENTS I thank the chair and members of my supervisory committee for their mentoring and the members of the Molecular Physiology Lab for their support and technical assistance. I thank Dr. Randy Braith and Kathy Howe for their collaborative efforts in this project. Also, I thank my family for their support and understanding as I completed my study. 3

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................3 LIST OF TABLES ...........................................................................................................................6 LIST OF FIGURES .........................................................................................................................7 ABSTRACT .....................................................................................................................................9 CHAPTER 1 INTRODUCTION..................................................................................................................11 Questions to be Addressed......................................................................................................13 Hypotheses..............................................................................................................................13 In Vivo.............................................................................................................................13 In Vitro.............................................................................................................................14 Significance............................................................................................................................14 2 REVIEW OF LITERATURE.................................................................................................15 Glucocorticoids and Muscle Remodeling...............................................................................15 Proteolytic Systems Involved in Skeletal Muscle Remodeling..............................................16 IGF-1 Pathway and Muscle Remodeling................................................................................19 Satellite Cells and Skeletal Muscle Remodeling....................................................................21 Nitric Oxide and Skeletal Muscle Remodeling......................................................................22 Glucocorticoids and Nitric Oxide...........................................................................................24 Concluding Statement.............................................................................................................25 3 MATERIALS AND METHODS...........................................................................................26 In Vitro Studies.......................................................................................................................26 Experimental Design.......................................................................................................26 Cell Culture.....................................................................................................................26 Histochemistry.................................................................................................................27 Nitric Oxide Production..................................................................................................28 Western Blots..................................................................................................................28 RNA Isolation and Quantitative Real-Time PCR...........................................................29 In Vivo Studies........................................................................................................................30 Experimental Design.......................................................................................................30 Protein Isolation and Western Blotting...........................................................................31 mRNA Measurements.....................................................................................................32 Single Fiber Isolation......................................................................................................32 Statistical Analysis..........................................................................................................34 4

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4 RESULTS...............................................................................................................................36 In Vitro Studies.......................................................................................................................36 MAFbx mRNA Expression.............................................................................................36 CAT and NOS mRNA Expression..................................................................................36 NOS and -Spectrin Protein Content..............................................................................36 Histological Measurements.............................................................................................36 Nitric Oxide Production..................................................................................................37 In Vivo Studies........................................................................................................................37 Body and Muscle Masses and Total Protein Content......................................................37 MAFbx, NOS, and CAT mRNA Expression..................................................................37 NOS Protein Content.......................................................................................................38 Satellite Cell Emanation..................................................................................................38 5 DISCUSSION.........................................................................................................................45 Acute Effects of Glucocorticoids............................................................................................45 Myotube Dimensions.......................................................................................................45 NO Availability...............................................................................................................46 Calpain Activation...........................................................................................................46 MAFbx Control...............................................................................................................47 Possible Mechanisms of Action for Nitric Oxide............................................................48 CAT Expression in Cultured Myotubes..........................................................................49 Chronic Effects of Glucocorticoids........................................................................................50 NOS Expression in Mouse Skeletal Muscle....................................................................50 CAT Expression in Mouse Skeletal Muscle....................................................................51 Satellite Cell Activity in Single Muscle Fibers...............................................................51 6 CONCLUSION.......................................................................................................................54 LIST OF REFERENCES...............................................................................................................55 BIOGRAPHICAL SKETCH.........................................................................................................66 5

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LIST OF TABLES Table page 3-1 Cell diagram illustrating experimental design of the cell culture portion of proposed project................................................................................................................................35 3-2 Cell diagram illustrating experimental design of the isolated myofiber portion of the proposed project.................................................................................................................35 4-1 mRNA data for MAFbx in control and dexamethasone treated cells................................39 4-2 mRNA data for CAT transporters in cell culture...............................................................39 4-3 Body and muscle masses and total protein content of animals..........................................39 4-4 mRNA data for CAT transporters in animals....................................................................39 6

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LIST OF FIGURES Figure page 4-1 Protein analysis -spectrin..............................................................................................40 4-2 Representative images from cells fixed in 3.9% formaldehyde and H & E stained..........41 4-3 Average area of individual myotubes................................................................................42 4-4 Nuclear number per myotube.............................................................................................42 4-5 Protein analysis nNOS and eNOS...................................................................................43 4-6 Satellite cells emanating from activated fibers..................................................................44 5-1 Illustration of the muscle maintenance pathways..............................................................53 5-2 Illustration of potential effects of nitric oxide...................................................................53 7

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LIST OF ABBREVIATIONS CAT: Cationic amino acid transporter MAFbx: Muscle Atrophy F-box NO: Nitric oxide eNOS: Endothelial nitric oxide synthase iNOS: Inducible nitric oxide synthase nNOS: Neuronal nitric oxide synthase 8

PAGE 9

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy GLUCOCORTICOID-INDUCED MYOPATHY IS MEDIATED BY IMPAIRED NITRIC OXIDE SYNTHESIS By Jodi Heather Dixon Long August 2007 Chair: David S. Criswell Major: Health and Human Performance Glucocorticoid drugs are potent and widely prescribed anti-inflammatory agents. However, glucocorticoid-induced skeletal muscle wasting severely limits the efficacy of these drugs, especially in chronic treatment situations. Understanding the mechanisms behind this muscle-wasting side effect will lead to more effective countermeasures, thus improving quality of life in various patient populations. The systemic effects of glucocorticoids are mediated, in part, by inhibition of inducible nitric oxide synthase (iNOS). Nitric oxide (NO) production by muscle serves important signaling functions. Therefore, NOS down-regula tion in skeletal muscle may contribute to glucocorticoid-related myopathy. Based on this, we hypothesized glucocor ticoid use inhibits NO production in skeletal muscle, and that this inhibition is partly responsible for the atrophy noted with glucocorticoid use. Furthermore, we hypothesized that addition of L-Arginine (L-Arg), a NOS substrate, and DETA-NONO, an NO donor w ould attenuate the glucocorticoid effects. We chronically (8wk) treated mice with prednisolone or saline at a concentration of 2.1mg/kg of body weight. At the end of the treatment period the animals were euthanised and their gastrocnemius removed for either Western Blot analysis or Single Fiber Isolation and culture. We demonstrated chronic exposure of mice to glucocorticoids decrease muscle satellite 9

PAGE 10

cell activity and NOS expression. In vitro treatment with the nitric oxide donor, DETA-NONO, restored satellite cell activity in myofibers isolated from glucocorticoid-treated mice to control levels. Additionally, we cultured L6 myotubes to study the acute effects of glucocorticoids. After 24h of treatment (100M dexamethasone), the myotubes displayed 6-fold increased expression of MAFbx, a muscle specific ubiquitin ligase. Co-treatment with DETANONO or L-Arg blunted the effect. Following 48h of treatment myotubes dimensions were assessed. The glucocorticoid treatment reduced myotube area by over 50%. When DETANONO or L-Arg was added, myotube atrophy was significantly attenuated. Thus, NO may be a useful therapeutic target for opposing the negative effects of both acute and chronic glucocorticoid use. 10

PAGE 11

CHAPTER 1 INTRODUCTION Muscle mass is determined by a delicate balance between protein synthesis and breakdown, which is keenly sensitive to active muscle tension and the pattern of muscle recruitment. Skeletal muscle atrophy, denoted by a decrease in muscle mass and fiber size, can arise from various etiologies including denervation, disuse, sepsis, cancer, AIDS, and chronic exposure to high levels of glucocorticoids. The latter could arise from adrenal hyperactivity (Cushings Disease) or pharmacological administration of glucocorticoids as anti-inflammatory agents. Regardless of the initiating factor, atrophy is characterized by a decrease in protein synthesis and an increase in protein degradation, with the latter having a larger impact. And, rather than simply an absence of growth or maintenance signals, muscle wasting is an active remodeling mechanism by which skeletal muscle myofibrils are dismantled and removed. Understanding this mechanism will lead to more effective strategies to attenuate muscle atrophy, thus improving quality of life in various patient populations. Glucocorticoid administration is linked to skeletal muscle atrophy (33, 34, 75) and increased expression of genes involved in proteolysis (17, 19, 37, 105). Additionally, the inhibition of calcium-activated proteases (calpains) in glucocorticoid treated myotubes attenuates the usual glucocorticoid-induced protein loss and decrease in myotube size (23). Therefore, glucocorticoids have the capacity to affect multiple components of the atrophy program. Three major proteolytic systems have been identified in skeletal muscle: the lysosomal, calpain, and ubiquitin-proteasome pathways. The lysosomal system is unlikely to be involved because it does not degrade myofibrillar proteins (24, 54, 111). It has been shown in multiple catabolic situations that contributions to atrophy via the lysosome are minimal (91, 99, 102). 11

PAGE 12

However, calpain and ubiquitin-proteasomal pathways seem to be intimately involved with the protein degradation associated with atrophy. The ubiquitin-proteasomal pathway is primarily responsible for degrading myofibrillar proteins into component amino acids. However, it does not degrade intact myofibrillar proteins (46, 84). Thus, myofibrillar loss initially is dependent on disassembly of myofibrils, which can occur via calpain activity. The dismantled myofibrils are then targeted to the proteasome for degradation, resulting in protein loss. Glucocorticoids activate both the calpain and the proteasome pathways (22, 23, 76, 100). Nitric oxide, a soluble gas product of L-arginine metabolism via nitric oxide synthase (NOS) activity, has rather global impacts on skeletal muscle. It is necessary for muscle differentiation (53), myoblast fusion (50, 67), satellite cell activation (2, 3, 93, 94), growth (78), and maintenance of muscle mass (44, 106). In fact, calpain activity, which initiates protein degradation, is inhibited by basal NO production (44). Attenuation of endogenous nitric oxide production in skeletal muscle may be an important mechanism of glucocorticoid-induced myopathy since NO production and expression of NOS enzymes are known to be downregulated in the presence of glucocorticoids (7, 41, 43). In preliminary studies, we have found a dramatic deficit in satellite cell number and responsiveness to mechanical activation in muscle fibers isolated from glucocorticoid-treated mice. Furthermore, 24h of treatment with DETA-NONO (an NO donor) can attenuate this satellite cell deficit (Betters et al., unpublished data). Therefore, the loss of NO production in skeletal muscle may initiate proteolysis via calpain and/or proteasome activation. Concomitantly, loss of NO may compromise the ability of skeletal muscle to maintain muscle mass in the long-term via a loss of satellite cell number and/or activity. 12

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Cellular availability of nitric oxide or its biochemical precursors may offer a convenient and effective target for treatment and prevention of glucocorticoid-induced myopathy. Therefore, this project will employ two experimental models, daily injection of prednisolone for 8 wks in adult mice and 24 or 48h treatment of cultured C2C12 mouse or L6 rat myotubes with dexamethasone, to examine the effects of glucocorticoid treatment on skeletal muscle NOS expression, calpain activity, expression of the muscle-specific ubiquitin E3 ligase, MAFbx, and abundance and activity of satellite cells. Further, treatment of cultured adult myofibers and L6 myotubes with the NO donor, DETA-NONO, and the NOS substrate, L-arginine will test the postulate that glucocorticoid-induced myopathies are related to compromised NO production. The potential for NO supplementation to ameliorate the negative effects of glucocorticoids on skeletal muscle will be assessed. Questions to be Addressed 1. What is the effect of glucocorticoid treatment on NOS protein expression and cationic amino acid transporter (CAT) mRNA expression in mouse skeletal muscle? 2. What is the effect of glucocorticoid treatment on NO production (nitrate accumulation), NOS protein expression, and CAT and MAFbx mRNA expression in myotubes? 3. What is the effect of glucocorticoid treatment on calpain activation? 4. What is the effect of glucocorticoid treatment on satellite cell activation? 5. Can nitric oxide supplementation in glucocorticoid treated myotubes inhibit MAFbx expression, calpain activity, and atrophy? 6. Can L-arginine supplementation ameliorate glucocorticoid-induced MAFbx expression, calpain activity, and atrophy in myotubes, due to enhanced nitric oxide production? Hypotheses In Vivo 1. Daily injections of prednisolone for 8 wks will decrease NOS protein and CAT mRNA expression in the gastrocnemius muscle of mice. 13

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2. Prednisolone treatment (8 wks) will reduce the number and activation/proliferation of satellite cells in single myofibers isolated from the mouse gastrocnemius muscle. 3. Treatment of isolated myofibers from prednisolone-treated mice with the NO donor, DETA-NONO, will increase the activation/proliferation of satellite cells in response to a mechanical stimulus. In Vitro 4. NO or L-arginine supplementation in dexamethasone-treated myotubes will increase NOS protein and CAT mRNA expression, limit MAFbx expression, and inhibit atrophy. 5. NO or L-arginine supplementation in dexamethasone-treated myotubes will decrease the abundance of calpain-specific proteolytic fragments of -spectrin. Significance The debilitating consequences of glucocorticoid-induced myopathies are of clinical relevance. Although the pathological overproduction of cortisol (i.e. Cushings Disease) is relatively rare, synthetic glucocorticoids are widely used pharmacologically for their dramatic anti-inflammatory effects. The loss of skeletal muscle can lead to loss of independence, falls or, potentially, respiratory failure. Thus, it is critical we understand the mechanisms that cause atrophy in response to chronic glucocorticoid treatment so that better countermeasures can be designed. Clearly nitric oxide is involved in normal skeletal muscle development, function, and hypertrophy (2, 67, 78, 83), but its role in attenuating atrophy is unclear. The attenuation of endogenous nitric oxide production due to glucocorticoid-induced loss of nNOS protein may represent an important signal initiating the atrophy process. If so, supplementation with the NOS substrate, L-arginine or with a pharmacological nitric oxide donor could be an effective strategy to ameliorate glucocorticoid-induced skeletal muscle myopathy. This study is designed to examine this possibility and contribute to our understanding of the mechanisms involved in muscle wasting. 14

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CHAPTER 2 REVIEW OF LITERATURE Skeletal muscle is a highly adaptive tissue. It is sensitive to not only changes in use, but also to various common pharmacological treatments. Muscle mass is determined by a delicate balance between protein synthesis and breakdown, and is capable of responding to increased or decreased loading by increasing (hypertrophy) or decreasing (atrophy) mass accordingly. Unfortunately, certain disease states (e.g. cancer, sepsis) and/or pharmacological agents (e.g. glucocorticoids) can induce rapid and severe muscle wasting. This skeletal muscle atrophy, denoted by a decrease in muscle mass and fiber size, is characterized by a decrease in protein synthesis and an increase in protein degradation, with the latter having a more prominent role. It is not wholly clear how glucocorticoids exert their effects, but their administration has been linked to increased expression of genes involved in proteolysis (17, 19, 37, 105). Therefore, delineating the mechanism of glucocorticoid-induced atrophy is key to developing countermeasures to this potentially debilitating condition. Glucocorticoids and Muscle Remodeling Glucocorticoids are a group of steroid hormones that have receptors throughout the body, thus their effects impact a number of physiological systems. The most widely understood effects are on carbohydrate metabolism and immune function. This class of hormones stimulates gluconeogenesis in the liver and increases amino acid mobilization from non-hepatic tissues. Additionally, they inhibit glucose uptake in muscle, and stimulate lipolysis in adipose tissue. Clearly these hormones elicit a catabolic effect in skeletal muscle. Despite the deleterious effects of chronic exposure to glucocorticoids, they are widely used pharmacologically for their anti-inflammatory and immunosuppressant qualities. 15

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Glucocorticoids have been shown to cause proteolysis in several situations (59). Treatment of C2C12 cells with dexamethasone, a synthetic glucocorticoid, not only inhibits proliferation (96), but also induces atrophy in terminally differentiated myotubes (7, 75, 89). In addition to protein degradation, dexamethasone administration induces MAFbx and MuRF1 expression (59, 75, 105). The up-regulation of these transcription factors was blocked by a pharmacologic inhibitor of glucocorticoids (113). Furthermore, glucocorticoid-induced MAFbx and MuRF1 expression seems to be operating through FOXO activation (76, 87), which is directly related to the PI3K/Akt pathway. In addition to the increase in components of the ubiquitin-proteasome pathway, dexamethasone increases intracellular concentrations of calcium in myocytes (Evenson et al. 2005, unpublished observation). This could in turn lead to increased calpain activity and may be one of many ways glucocorticoids exert their wasting effects on skeletal muscle. Another potential path is via inhibition of IGF-1 signaling. In the absence of IGF-1, Akt is dephosphorylated which leads to FOXO activation (76), thus promoting atrophy. Additionally, it should be noted that diminished Akt signaling promotes the cleavage and activation of caspase-3 (85). IGF-1 has been shown to attenuate the effects of glucocorticoids on L6 cells (52). In fact, Latres et al. (48) demonstrated an inverse regulation of MAFbx dependent on IGF-1 presence and determined that both the Akt/FOXO and Akt/mTOR pathways are needed for transcriptional changes induced by IGF-1. Proteolytic Systems Involved in Skeletal Muscle Remodeling Three major proteolytic systems have been identified in skeletal muscle: the lysosomal, calpain, and ubiquitin-proteasome pathways. The lysosomal system is unlikely to be involved in atrophy because it does not degrade myofibrillar proteins (24, 54, 111). It has been shown in multiple catabolic situations that contributions to atrophy via the lysosome are minimal (91, 100, 16

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102). However, calpain and ubiquitin-proteasomal pathways seem to be intimately involved with the protein degradation associated with atrophy. The calpains (calcium-activated proteases) are comprised of a family having at least 14 members. Some of these are ubiquitous enzymes, such as and m-calpain, while others are tissue specific proteins, such as the muscle specific calpain 3, also called p94 (28, 86, 90). The and m-calpain isoforms are named based on the calcium concentration needed to attain their half-maximal proteolytic activation. The -calpain form requires M concentrations and the m-calpain requires mM concentrations (5, 16, 20). Both levels of calcium are supraphysiological (16, 55), so some mechanism exists to activate the normally inactive calpains. This is evident in that calpains are relevant in physiological systems (55), as demonstrated by the work showing that in vivo calpain inhibition has a protective effect on skeletal muscle (70, 101). The calpains are activated in skeletal muscle during reduced use (79, 91). When activated these proteases target structural proteins within the muscle cell such as talin, a linker protein in the cell membrane (44). The dismantling of the cytoskeleton via calpain activation seems to be a critical event in cellular protein degradation (27, 39). The regulation of calpain activity is complex. The most important activator of calpain is calcium (28). Cytoplasmic calcium is increased in glucocorticoid treated cell cultures (8, 48, 103). Furthermore, Wei et al. (109) has shown that calpain inhibitor (calpeptin) administration blocks dexamethasone-induced proteolysis. Thus, one potential role dexamethasone plays in the atrophic response is to increase cytosolic calcium with subsequent calpain activation. Calcium is not the only regulator of calpain-mediated proteolysis. Calpastatin (an endogenous inhibitor of calpains) and nitric oxide may play a regulatory role (44). Calpastatin prevents calpain enzymatic activation and the expression of catalytic activity (27). Nitric oxide 17

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has been shown to regulate sarcoplasmic reticulum function and therefore intracellular calcium. This action inhibited calpain action in ischemia-reperfused rat hearts (14). Zhang, Kraus, and Truskey (114) showed NO inhibited calpain-mediated proteolysis of talin in C2C12 myotubes. Thus, calpains are regulated by a number of factors. Calpain activity is likely not the sole contributor to atrophy in the presence of elevated glucocorticoids. The role of the ubiquitin-proteasome pathway in atrophy cannot be discounted. The cellular events involved in this method of protein breakdown are highly coordinated. First, the protein to be degraded is marked by attaching ubiquitin to it. This is accomplished via a trifecta of proteins: E1, the ubiquitin-activating enzyme, E2, the ubiquitin-conjugating enzyme, and E3, the ubiquitin-protein ligases (e.g., MAFbx or atrogin-1 and MURF1) (40). E1 activates ubiquitin and transfers it to E2. Then, E3 transfers the ubiquitin from E2 to the target protein. This occurs multiple times until the protein to be degraded is marked by a chain of ubiquitin molecules. Once polyubiquinated, the targeted protein is rapidly degraded by the proteasome (26, 58, 98). Atrophy is partly a result of the ubiquitin-proteasome pathway. In both the skeletal muscle of immobilized rat hindlimb and atrophied cardiac muscle, mRNA for MAFbx and MuRF1 were increased (47, 71). Ikemoto et al. (40) demonstrated an increase in ubiquinated proteins within skeletal muscle during reduced use. There is a concomitant increase in proteasome enzyme activity (79). It has been demonstrated by a number of authors that this system accounts for most of the elevated muscle proteolysis in a variety of atrophic situations (36, 91, 95) including glucocorticoid use (38, 112). 18

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IGF-1 Pathway and Muscle Remodeling IGF-1 (insulin-like growth factor-1) is a growth factor that initiates a cascade of intracellular signaling events leading to hypertrophic and antiproteolytic responses. Two main pathways stimulated by IGF-1 are the Ras-Raf-MEK-ERK pathway and the PI3K/Akt pathway. The Ras-Raf-MEK-ERK signaling events affect fiber type composition, but not myofiber size (62), thus this pathway is likely unimportant in causing hypertrophy or atrophy. However, the PI3K/Akt pathway seems to cause muscle hypertrophy ultimately by stimulating translational events via the mTOR and GSK kinases (12). mTOR, one target of Akt, when phosphorylated will activate p70S6 kinase. This kinase increases muscle protein translation, ultimately supporting hypertrophy. Recently Thompson and Gordon (97) demonstrated that older rats subjected to skeletal muscle overload had less phosphorylation of mTOR and its downstream target p70S6K that correlated with diminished total protein content. Further evidence of a hypertrophic role of mTOR was shown by rapamycin blocking myotube hypertrophy in a C2C12 model of terminally differentiated myotubes (63, 65). The Park et al. group further demonstrated the necessity of mTOR for hypertrophy by stably expressing in C2C12s rapamycin-resistant forms of mTOR and p70S6K. The rapamycin-resistant cultures underwent hypertrophy with rapamycin treatment, while the control cultures did not hypertrophy. This was assessed by image analysis. An additional role mTOR plays in muscle remodeling is through inhibition of FOXO (forkhead box O), which targets at least one of the E3 ligases. Based on the evidence presented here, mTOR, when phosphorylated by Akt will cause hypertrophy and inhibit atrophy (25, 49). Akt also directly targets the forkhead box O (FOXO) family of transcription factors, which target at least one of the E3 ligases. Phosphorylated Akt inactivates (phosphorylates) FOXO, thus hindering protein degradation. FOXO, when active, translocates to the nucleus and 19

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increases transcription of a key element of the ubiquitin-proteasome pathway, the ubiquitin ligase atrogin-1 or MAFbx. The functional importance of this protein being expressed during the atrophy process was demonstrated by using MAFbx/ mice. Mice lacking the MAFbx gene had less atrophy following denervation (11). This was the same for the other E3, MuRF1 in a MuRF1-/knockout mouse model. Furthermore, because MuRF1 and MAFbx have been shown to be upregulated in other models of atrophy, they are widely accepted as reliable markers of the atrophy process (11, 12, 29, 51, 113). So, under the influence of IGF-1 through Akt signaling, the activity of FOXO is repressed while that of mTOR is increased (12). This causes an increase in cell size through a concomitant increase in protein synthesis and a decrease in protein degradation. Alternatively, in the absence of IGF-1, there is an increase in proteolysis and the expression of atrophy-related ubiquitin ligases such as MAFbx, correlated with FOXO dephosphorylation (activation) (76). FOXO factors are critical in the transcription of MAFbx in myotubes under both starvation and glucocorticoid treated conditions (76). In both situations, IGF-1 signaling decreases. Thus phosphorylation of FOXO via Akt activation is minimized (13, 64, 92), and FOXO translocates to the nucleus (9, 13) to up-regulate MAFbx expression (76), and hence induce atrophy. Further support for the necessity of nuclear FOXO in atrophic conditions was established when a constitutively active form of FOXO3 (one of the three mammalian FOXOs associated with skeletal muscle) remained in the nucleus and caused atrophy (76, 82, 88). In addition to Akt regulating protein balance in skeletal muscle via FOXO and mTOR, it also targets glycogen synthase kinase (GSK3). GSK3 is blocked by phosphorylated (active) Akt, and leads to hypertrophy (72). However, when GSK3 is activated, it blocks transcriptional initiation factors necessary for protein synthesis (12). This would indicate GSK3 is a potential 20

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mitigator of skeletal muscle atrophy. However, Sandri et al. (76) demonstrated that activation of GSK3 does not influence the induction of MAFbx. So, perhaps GSK3s role in muscle metabolism is limited to its control of initiation factors involved in protein synthesis. The majority of the evidence regarding atrophy in skeletal muscle indicates that down-regulation of IGF-1 signaling is a key event leading to proteolysis. There seem to be two parts to the atrophic response via this mechanism. First, definitive links have been established regarding nuclear localization of FOXO factors in the absence of IGF-1 signaling and also FOXO factors binding directly to the MAFbx promoter to cause an increase in this ubiquitin ligase (76). Second, Song et al. (85) showed that down-regulation of IGF-1 signaling via the Akt/mTOR pathway leads to calpain activation. It is fairly widely recognized that calpains likely dismantle the existing cytoskeleton, readying targeted myoproteins for ubiquination and subsequent degradation via the proteosome. Satellite Cells and Skeletal Muscle Remodeling Adult skeletal muscle fibers consist of post-mitotic, terminally differentiated nuclei. Each of these nuclei govern a finite volume of cytoplasm, the size of which seems to be set and tightly regulated (i.e. the nuclear domain hypothesis) (31). In order for skeletal muscle fibers to recover from injuries or adapt their size to meet functional demands, and still maintain this nuclear domain range, a source of nascent myonuclei must be available. Muscle satellite cells, which are quiescent muscle precursor cells found between the myofibers and the external lamina, provide this source. In response to growth or regeneration stimuli, these cells are activated to differentiate and join an existing myofiber or form a new myotube, thus maintaining the myonuclear domain. 21

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Activation of satellite cells is a necessary step in adult skeletal muscle growth. Several studies have used localized gamma irradiation of rat skeletal muscles to eliminate mitotically active cells within the muscle, without compromising circulating stem cells or growth factors (66, 73). These irradiated muscles are completely incapable of regaining muscle mass following injury (60, 73) or growing in response to mechanical overload (1, 73). This suggests that addition of nascent myonuclei is required for muscle growth rather than simply following changes in fiber size. In addition to supporting muscle growth and regeneration, satellite cells may be important for maintenance of muscle mass. Atrophied muscles from hindlimb suspended animals contain fewer satellite cells associated with isolated myofibers (61). It seems that repeated use of myogenic precursor cells for muscle repair can lead to exhaustion of the satellite cell pool (18, 108). Additionally, it appears that these same satellite cells from atrophied animals have an impaired ability to activate and proliferate (61). As myonuclei are removed during atrophy, the down-regulation of satellite cell number and activity likely prevents the addition of new nuclei to maintain the fiber size. Perhaps this de-activation of satellite cells is a primary mechanism to inhibit hypertrophy and induce atrophy. Nitric Oxide and Skeletal Muscle Remodeling Nitric Oxide (NO), a product of L-arginine (L-Arg) metabolism, is a free radical that is produced by nitric oxide synthase (NOS) enzymes. All three isoforms of the NOS enzyme can be expressed in skeletal muscle. nNOS is most abundant, being associated with the dystrophin complex and localized in costameres at the sarcolemma (30). eNOS is also constitutively expressed in muscle and may be associated with mitochondria (42). Lastly, iNOS is believed to be only expressed in response to an inflammatory stimulus (87). Although NO is primarily known for its vasodilatory effects, it is also an important regulatory molecule in many different 22

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tissues, including skeletal muscle (87). Our lab has demonstrated that nitric oxide (NO) positively influences skeletal muscle hypertrophy and contractile gene expression during overload (78, 83). Anderson (2) found that NO is a primary signal for skeletal muscle satellite cell activation. Others have shown nitric oxide to be important in myoblast fusion (50, 67). Thus, it is clear that NO is involved with the hypertrophic response. NO production increases in isolated rat glomeruli when incubated with IGF-1 (107). Additionally, an increase in Akt phosphorylation was noted. This rise in Akt phosphorylation associated with NO presence has also been demonstrated in bovine aortic endothelial cells (56). Furthermore, when an mTOR inhibitor was used in activated macrophages, NO production was reduced (110). While these studies are not in skeletal muscle, they demonstrate a potential link between NO production and the PI3K/Akt/mTOR signaling pathway. In addition to nitric oxides influence on or production from hypertrophic pathways, it has also been associated with components of the atrophic pathway. For example, Koh and Tidball (44) showed evidence that NO could be a regulatory molecule of calpains. They used sodium nitroprusside, an NO donor, and showed no proteolysis of structural proteins. Moreover, through zymography and an activity assay, inhibition of m-calpain in C2C12 cultures was found. Michetti et al. (57) also showed inhibition of m-calpain in a dose-dependent manner with sodium nitroprusside. Further support for a role of NO in calpain inhibition comes from Chohan et al. (14). They demonstrated that ischemia-reperfused hearts had an increase in calpain activity and a decrease in cytosolic NO levels. When L-Arg was administered, the increased calpain activity was attenuated. Thus, whether NO was directly given or synthesized via one of the NOS enzymes and L-Arg, it inhibits calpain activity, suggesting that some basal endogenous NO production may inhibit proteolysis and support maintenance of muscle mass. 23

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Endogenous NO production is dependent upon L-Arg uptake (80). L-Arg is transported into the cells via a sodium-independent y+ transport system. This system recognizes cationic amino acids, specifically L-Arg, L-Lys, and ornithine (15). There are three known transporters for L-Arg present in skeletal muscle: CAT-1, CAT-2A, CAT-2B. CAT-1 and CAT-2B have a high affinity for L-Arg compared to CAT-2A. There is some evidence that NO production in skeletal muscle may be limited by L-Arg availability. Since plasma levels of L-Arg generally exceed the Km value for the NOS enzymes, an L-Arg limitation would implicate the CAT transporters as potential regulators of NO production. Glucocorticoids and Nitric Oxide Glucocorticoids, known for their atrophy-inducing qualities, suppress NO production (41, 80). In fact, much of the anti-inflammatory actions of glucocorticoids are likely due to inhibition of iNOS. Dexamethasone inhibits iNOS transcription and mRNA expression (6, 45). Further, glucocorticoids have been shown to induce proteolytic cleavage of iNOS at a specific site within the calmodulin binding domain via activation of calpain (103). This domain is conserved among the three NOS isoforms, raising the interesting possibility that glucocorticoids may globally down-regulate NO production, simultaneously compromising the signaling actions of nNOS and/or eNOS while inhibiting the inflammatory effects of iNOS. In cardiac microvascular endothelial cells, treatment with glucocorticoids completely abolishes NO production, an effect attributed to eNOS down-regulation (80). eNOS has been shown to be down-regulated by FOXO1 and FOXO3A (68). These factors bind to the eNOS promoter in endothelial cells. This causes repression of eNOS expression. Glucocorticoids may also affect NO production via L-Arg availability. Simmons et al. (80) showed a reduction in L-Arg uptake in dexamethasone treated cells. Dexamethasone treatment prevented the induction of CAT-2B mRNA by cytokines. Furthermore, Hammermann 24

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et al. (32) found a down-regulation of CAT-2B in rat alveolar macrophages after 20h exposure to dexamethasone. It seems that the inflammatory response increases L-Arg transport into the cells, which supports large-scale NO production by iNOS. Dexamethasone, however, is able to not only attenuate this increase in L-Arg influx by the down-regulation of CAT transporters, but also seems to suppress iNOS (81). Therefore, glucocorticoids have the ability to greatly reduce NO production by inhibiting both substrate availability and NOS enzyme expression. Given the importance of NO signaling for skeletal muscle remodeling as discussed above, inhibition of endogenous NO production may be an important mechanism for glucocorticoid-induced skeletal muscle myopathy. Concluding Statement Our lab has demonstrated that nitric oxide (NO) positively influences skeletal muscle hypertrophy. Koh and Tidball (44) found that NO inhibition of calpains protected cells from proteolysis. Wang et al. (106) inhibited NO production via L-NAME administration and induced atrophy. Moreover, glucocorticoid use can decrease NO production, increase intracellular concentrations of calcium and induce expression of MAFbx. Taken together, it is not unreasonable to believe that glucocorticoid-induced skeletal muscle myopathy is due, at least in part, to compromised NO production. An NO-donor and/or supplementation with the NOS substrate L-Arg could attenuate glucocorticoid-induced atrophy. This study explored the effects of glucocorticoid treatment on NOS and CAT expression in skeletal muscle using both in vivo and in vitro models. Further, the potential for an NO donor or L-Arg supplementation to inhibit muscle atrophy via down-regulation of MAFbx expression and calpain activity, and improved satellite cell function was experimentally tested. 25

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CHAPTER 3 MATERIALS AND METHODS The purpose of this project was to examine the mechanism of glucocorticoid-induced skeletal muscle myopathy, and test the hypothesis that the loss of satellite cell activity and initiation of the calpain and proteasome proteolytic pathways during exposure to glucocorticoids is mediated by the loss of endogenous nitric oxide production. In Vitro Studies Experimental Design The C2C12 mouse and L6 rat myogenic cell lines were used to examine the effects of exposure to the glucocorticoid, dexamethasone. Differentiated myotube cultures were treated with dexamethasone for 48h and monitored for myotube dimensions, protein content, and proteolytic cleavage of the calpain substrate, -spectrin. Alternatively cultures were treated for 24h and assessed for expression of the ubiquitin ligase MAFbx (or atrogin-1) and nitric oxide production. Supplementation of cultures with DETA-NONO, a nitric oxide donor, or L-arginine, the NOS substrate, were performed to test for a causal relationship between compromised nitric oxide production and initiation of the atrophy program. Table 3-1 illustrates the design of this study. Cell Culture C2C12 and L6 cells (ATCC, Manassas, VA) were plated and proliferated in Dulbeccos Modified Eagles Medium (DMEM) growth media (GM) containing 10% FBS and 1% penicillin/ streptomycin. At 80% confluency the GM was removed and the cells washed twice with 37C PBS. Then differentiation media (DM) was added (DMEM supplemented with 2% horse serum and 1% penicillin/ streptomycin). Cultures differentiated for 5-7 days until there were confluent myotubes. At that point cultures were divided into 3 groups and treated for 24h 26

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or 48h with one of the following: 10 mM L-arginine, 10M DETA-NONO, or no supplement (control) in DM. Half of the cultures from each group were co-treated with 100M dexamethasone. At the time of harvest, cultures were rinsed twice with ice cold PBS and then harvested in two manners. For protein analysis via Western Blots, the cells treated for 48h were harvested on ice in 0.300ml non-denaturing lysis buffer (NDL: 20% Triton X-100; 1M Tris/ pH=7.5; 5M NaCl; 0.5M EDTA; 10mg/ml sodium azide; 4mg/ml NaF; 4ug/ml NaVO 3 ; 0.1% protease inhibitors) and then centrifuged at 500 x g for 5min to remove insoluble material. The supernatant was used for Western Blots for nNOS, eNOS, and -spectrin. For mRNA analysis, parallel cultures treated for 24h were harvested in 1ml of ice cold TRIzol (Invitrogen, Carlsbad, CA). Histochemistry Parallel cultures were fixed with 3.7% formaldehyde and stained with hematoxylin and eosin at 48h. Microphotometric digital images of each culture were captured using a Zeiss Axiovert200 light microscope (Thornwood, NY) and Qimaging RETIGA EXi digital camera (Surry, BC, Canada) and software (IPLab3.6.5, Scanalytics, Rockville, MD). The images were evaluated for myotube dimensions (area and nuclei/ area) and density (myotube number/ field of view) using Scion Image imaging software. Three images of each culture were captured. Within each image 10 myotubes were analyzed for area and nuclear content by measuring the first 10 myotubes in each field of view beginning in the left-hand corner. Thus, 30 myotubes were analyzed per culture and each treatment had three cultures for a total n = 90 myotubes per treatment. 27

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Nitric Oxide Production Nitric oxide production by the cells was measured by assessing nitrate accumulation in media measured at 0h, 24h, and 48h. Following collection, the media was centrifuged at 5000 x g for 40min using microcentrifuge filters to remove serum, which can interfere with the kit components. The Nitrate/Nitrite Fluorometric Assay Kit from Cayman Chemical Company (Ann Arbor, MI) based on the procedures described by Misko et al. (1993) was used. Western Blots Myotube protein was collected as described above. Protein concentration was determined by DC assay (Bio-Rad, Rockville Centre, NY). SDS-PAGE was performed on 7% polyacrylamide gels. Equal amounts of protein were loaded into each lane. Positive controls for nNOS and eNOS were used. The gels were run at 60V for 1h and then 100V for 1h (NOS blots) or 100V for 2h (-spectrin blots) with a 1h transfer at 500mA. The transfer was onto nitrocellulose membranes that were subsequently blocked in Odyssey blocking buffer for 1h. The membranes were then incubated overnight with primary antibody in 1:1 TBS-T (0.01%): Odyssey blocking buffer. For nNOS and eNOS, a 1:500 dilution was used (monoclonal, mouse, anti-nNOS: 611852, eNOS: 610328, BD Transduction Laboratories, San Jose, CA). For -spectrin (Biomol), a 1:5000 dilution was used. -actin incubation was used as a loading control (1:4000 dilution). Following the overnight incubation at 4C, the membranes were washed 4 times for 5min each with TBS-T. The fluorescent secondary antibody (anti-mouse HRP, Amersham, Piscataway, NJ) was applied to the -spectrin membrane using a 1:3000 dilution, while the nNOS blot was incubated with anti-rabbit using a 1:8000 dilution. All blots were then incubated at room temperature for 35min. Then membranes were washed four times for 5min each with TBS-T and two additional washes with TBS. The membranes were scanned on the 28

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Odyssey infrared imaging system (Li-Cor, Lincoln, Nebraska) and the relative fluorescence of the bands was quantified by densitometry using the accompanying software. Results are expressed as mean SEM. RNA Isolation and Quantitative Real-Time PCR We examined expression of mRNA transcripts for iNOS, the two cationic amino acid transporters responsible for L-Arg uptake in skeletal muscle cells (CAT-1 and CAT-2), and MAFbx. Total RNA was extracted from cultured cells by harvesting in 1 ml of TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturers instructions. Concentration and purity of the extracted RNA was measured spectrophotometrically at A 260 and A 280 in 1X TE buffer (Promega, Madison, WI). Purified RNA was then stored at -80 C for later assay. Reverse transcription (RT) was performed using the SuperScript III First-Strand Synthesis System for reverse transcription-polymerase chain reaction (RT-PCR) according to the manufacturers instructions (Life Technologies, Carlsbad, CA). Reactions were carried out using 1 g of total RNA and 2.5 M oligo(dT) 20 primers. First strand cDNA was treated with two units of RNase H and stored at -80 C. Primers and probes for CAT-1, CAT-2, iNOS, and MAFbx (assay # Mm00432019_m1, Mm00432032_m1, Mm00440485_m1, and Rn00591730, respectively) were obtained from the ABI Assays-on-Demand service and consisted of Taqman 5' labeled FAM reporters and 3' nonfluorescent quenchers. Primer and probe sequences from this service are proprietary and therefore, are not reported. Primer and probe sequences also consisting of Taqman 5' labeled FAM reporters and 3' nonfluorescent quenchers for hypoxanthine guanine phosphoribosyl transferase (HPRT) obtained from Applied Biosystems (Assays-by-Design) are: Forward, 5'29

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GTTGGATACAGGCCAGACTTTGT-3'; Reverse, 5'-AGTCAAGGGCATATCCAACAACAA -3'; Probe, 5'-ACTTGTCTGGAATTTCA-3'. Quantitative real-time PCR was performed using the ABI Prism 7700 Sequence Detection System (ABI, Foster City, CA). Each 25 l PCR reaction, performed in duplicate, contained 1 l of cDNA reaction mixture. In this technique, amplification of the fluorescently labeled probe sequence located between the PCR primers was monitored in real-time during the PCR program. The number of PCR cycles required to reach a pre-determined threshold of fluorescence (called the CT) was determined for each sample. Samples were quantified relative to the CT for a normalizing gene determined separately in the same sample. This procedure is referred to as the comparative CT method as described by Bustin (2002). HPRT was selected as the appropriate normalizer since the expression of this gene in C2C12 cells is not significantly altered during differentiation and fusion (p > 0.05). In Vivo Studies Experimental Design Adult mice were treated with the glucocorticoid, prednisolone, for 8 wks to examine the effects on skeletal muscle mass, protein content, satellite cell abundance and activity, and the potential for a nitric oxide donor to rescue prednisolone-induced deficits in satellite cell activation/proliferation in isolated, cultured skeletal muscle fibers. Animals. Seven-month old male Swiss-Webster mice were obtained from Harlan (N=16). After a seven-day acclimation period, mice were weighed and then given a subcutaneous injection daily, six days per week in the morning for eight weeks. They received either Depo-Medrol (prednisolone; Pharmacia & Upjohn, Kalamazo, MI; n=8) or sterile saline (n=8) at a concentration of 2.1mg/kg of body weight. All injections were prepared so that each injection was approximately a 0.1ml volume. Food (normal mouse chow given ad libitum) was weighed 30

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every three days for the duration of the study to account for weight loss due to reduced food consumption. Animals were weighed weekly on the non-injection day, and treatment dosage was adjusted for the next week based on the current animal weight. They were housed one to a cage in SPF-2 at ACS, University of Florida, under normal conditions (12h light/dark cycle). On the last day of the study they were anesthetized with inhaled isoflurane (2-3.5%) with oxygen as the carrier gas using an anesthesia cart (with the charcoal filter scavenger attached). Euthanasia was by exsanguination and was confirmed with cervical dislocation. Following this, the animals skin and superficial fascia of the hind limbs was removed to expose the muscles of the hind limbs. The gastrocnemius (GN) muscle was dissected out and weighed immediately. In four animals per group, the muscle was flash frozen in liquid nitrogen for subsequent analysis of muscle protein content and Western blot procedures. The gastrocnemius from the remaining four animals from each group was digested with collagenase for myofiber isolation. (IACUC protocol #E401) Protein Isolation and Western Blotting Frozen muscles were thawed and homogenized in buffer (0.1M Tris Base; 0.1M NaPO 4 ; 0.01M EDTA; 30% glycerol; pH=7.8) with protease inhibitors (200mM Benzamidine; 1mg/ml Pepstatin A and Aprotinin; 5mg/ml Leupeptin; 1M DTT; 40mM phenylmethylsulfonyl) at a 1:5 dilution based on muscle weight. This was done on ice, with the dismemberator on for two bouts of 10sec each. Following homogenization, the slurry was allowed to sit on ice for 10min. Then 100ul was removed and added to 700ul of 0.05M NaOH and left overnight at room temperature for total protein versus connective tissue assay. The remainder of the slurry was centrifuged at 750 x g for 15min at 4C. The supernatant was transferred to clean microcentrifuge tubes and stored at -80C until used for Western Blotting. 31

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Total protein and connective tissue protein was measured in the homogenized samples. Following an overnight digest (at least 18h) in 0.05M NaOH, total protein was assessed using the DC assay (Bio-Rad) and then the samples were centrifuged at 4000 x g for 15min to pellet the connective tissue and then the supernatant was subjected to the protein assay again. The second assessment of protein content is considered to be the protein in the non-connective tissue fraction. This measurement was used to determine equal protein loading for the Western Blots. Western blot analysis for nNOS and eNOS in chronically treated muscle was done by methods described previously but the protein was transferred to a PVDF membrane. These membranes were blocked in 5% non-fat dry milk in TBS-T (0.01%) for 1h. They were rinsed in TBS-T and incubated overnight at a 1:500 dilution (polyclonal anti-nNOS, Cayman Chemical, Ann Arbor, MI; polyclonal anti-eNOS BD Transduction Laboratories, San Jose, CA). Secondary antibodies (anti-rabbit and anti-mouse for nNOS and eNOS, respectively; Sigma, St. Louis, MO) were applied for 1h (1:5000). Protein bands were detected with ECL+ (Amersham Biosciences, UK). The relative amounts of the bands were quantified by densiometry using ImageJ software. mRNA Measurements Total RNA was isolated from gastrocnemius (GN) samples after homogenization in Trizol. Reverse transcription followed by quantification of iNOS, CAT-1, and CAT-2 mRNA by quantitative real-time PCR was performed as described above for the cell culture experiment. Single Fiber Isolation GN muscles (N = 4 animals/group) were surgically removed by first removing the skin of the hindlimbs, exposing the Achilles tendon. Then a small incision was made behind the knee and forceps and small sharp scissors were used to cut away the upper layer of fascia and connective tissue down the length of the muscle. As much as possible of the fascia was carefully peeled away using a scalpel. Then the tendons at the knee were cut on either side at an angle. 32

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Without pulling on the muscle, the Achilles tendon was cut and the GN pulled up while carefully trimming connective tissue on either side. This was done on both limbs. Once the GNs were removed, they were rinsed in PBS, filleted, and then immediately transferred to a 15ml falcon tube containing digest media (9ml DMEM, 1% penicillin/ streptomycin, 1ml collagenase). The digest was rocked at 37C for 1.5h. Following the digest, the muscles were titurated three times with a wide-bore 10ml disposable pipet, then the fibers were allowed to settle. After that, the digest media was removed with a pipet down to about the 3ml mark. Then about 10ml of fresh, pre-warmed media (DMEM, 10% FBS, 1% penicillin/ streptomycin) was added to the falcon tube containing the fibers, and the tube inverted a few times. The media was removed again and the rinse repeated 1-2 more times. This diluted the collagenase. At this point 3-4ml of the digest was transferred to a series of p100 plates containing warm media. Quality fibers were removed from the surrounding dead (hypercontracted) fibers and debris by transferring to a clean plate (containing 15ml media) using a wide-bore 200l pipet. Plates were not out of the incubator longer than 10min during this process. Once the chosen fibers were ready to plate, 1ml of warm media was added to each well of a 24-well plate and the plate was allowed to equilibrate in the incubator for 10-15min. Then a drop of matrigel was applied to each well. The fiber was added by extracting them from the clean plates in 50l volume using a p200 pipet tip (with the end snipped-off) and another drop of matrigel was applied. After all fibers were plated, the 24-well plate was placed in the incubator for at least 15min. Then the plate was centrifuged at 1100 x g at 37C for 30-40min to activate the satellite cells. The fibers were maintained at 37C, 5% CO 2 for 24h. Treatments of DETA-NONO (0, 5, 10, 50M) were added to the fibers from both control and glucocorticoid treated animals following the centrifugation step. After 24h, the cultures were fixed in 2% formaldehyde and immunohistochemistry performed for MyoD and 33

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DAPI staining. The number of myogenic cells was quantified to assess activation or proliferation of satellite cells. Table 3-2 illustrates this design. Statistical Analysis Statistical analyses were performed on SPSS (version 12.0.1) using 2-way ANOVAs (glucocorticoid vs. supplement). Post hoc analyses were performed using Tukeys test. Significance was established at p<0.05. 34

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Table 3-1. Cell diagram illustrating experimental design of the cell culture portion of proposed project. Dexamethasone (100 M) + No Supplement Control n=6 n=6 L-Arginine (10 mM) n=6 n=6 DETA-NONO (10 M) n=6 n=6 Table 3-2. Cell diagram illustrating experimental design of the isolated myofiber portion of the proposed project. Fibers isolated from Control mice (n=4 mice) Fibers isolated from Prednisolone-treated mice (n=4 mice) No Supplement Control ~100 fibers ~100 fibers 5 M DETA-NONO ~100 fibers ~100 fibers 10 M DETA-NONO ~100 fibers ~100 fibers 50 M DETA-NONO ~100 fibers ~100 fibers 35

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CHAPTER 4 RESULTS In Vitro Studies MAFbx mRNA Expression Dexamethasone treatment of L6 myotubes resulted in a six-fold increase in MAFbx mRNA expression compared to control (Table 4-1). Supplementation of arginine (10mM) or DETA-NONO (5M) significantly blunted the effect of the dexamethasone, even though MAFbx mRNA did not reach control levels (Table 4-1). Treatment of the L6 myotubes with arginine alone significantly reduced MAFbx expression, whereas DETA-NONO supplementation alone showed a trend toward lower MAFbx expression but did not reach statistical significance (Table 4-1). The interaction between treatment groups was significant at all levels (p<0.05). Thus, the upregulation of MAFbx seen with dexamethasone use may be attenuated by L-Arg or DETA-NONO. CAT and NOS mRNA Expression In C2C12 cells, the mRNA expression for both transporters (CAT-1 and CAT-2) was significantly depressed in glucocorticoid treated cells (p<0.05) (Table 4-2). CT values for iNOS were below detectable limits. NOS and -Spectrin Protein Content There was no significant difference in the cleaved/total -spectrin protein expression normalized to -actin with any treatment in cell culture (p=0.818) (Figure 4-1). nNOS and eNOS expression in both L6s and C2C12s was not detectable. Histological Measurements Size, nuclear number, and total myotube area per field of view were decreased in glucocorticoid-treated cells (Figure 4-2). The total myotube area per field of view dropped by 36

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26% after exposure to dexamethasone, and this effect was attenuated with arginine treatment (only an 8.1% decrease when L-Arg was added). The dexamethasone effect was more moderate when DETA-NONO was added (23% decrease). Not only was less of the field of view covered by myotubes in the glucocorticoid-treated cells, these myotubes were also smaller (64%). When DETA-NONO or L-Arg was added to the dexamethasone-treated cells, the myotubes were only 42% smaller than controls (Figure 4-3). Nuclear number was significantly decreased by dexamethasone treatment (p<0.05), and this was attenuated when L-Arg or DETA-NONO was added to the medium (Figure 4-4). While confirming dexamethasone causes atrophy, these data also indicate that NO may partially attenuate the loss in size and nuclear number. Nitric Oxide Production Nitric oxide production was measured in vitro via 24h nitrate accumulation in the media. Media was assessed at time 0h, 24h, and 48h. Regardless of the treatment or time point, there were no differences between groups for fluorescence. So, cells treated with dexamethasone may not have compromised NOS activity. In Vivo Studies Body and Muscle Masses and Total Protein Content Body mass and gastrocnemius mass did not significantly decline with 8 weeks of prednisolone treatment (Table 4-3). Total protein/gastrocnemius muscle did not significantly decrease, but there was a trend to be lower in glucocorticoid muscle compared to control. MAFbx, NOS, and CAT mRNA Expression MAFbx mRNA levels are expected to have reached a steady state of expression after 8 weeks of glucocorticoid treatment, thus this was not analyzed in muscle homogenate. However, CAT and iNOS expression were considered. CAT expression was insignificantly elevated in the 37

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glucocorticoid treated animals (Table 4-4). iNOS values were below detectable limits, and thus not reported. NOS Protein Content The expression of nNOS and eNOS protein was reduced by over 30% in the glucocorticoid treated animals compared to controls (Figure 4-5B). Representative blots are displayed in Figure 4-5A. Satellite Cell Emanation MyoD+ satellite cells emanating from isolated myofibers 48 hours after centrifugation were significantly reduced with in vivo prednisolone treatment (54% of control fibers had emanating cells present vs. 25% of glucocorticoid fibers, Figure 4-6). However, supplementation of culture media with DETA-NONO (5-50 M) induced satellite cell emanation in a dose-dependent manner. Fifty M DETA-NONO eliminated the difference in centrifuge-induced satellite cell emanation between glucocorticoid and control fibers (76% of control vs. 74% of glucocorticoid). This suggests that NO bioavailability limits satellite cell activity in response to a mechanical stress. Further, exogenous NO can rescue compromised satellite cell function following glucocorticoid treatment. 38

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Table 4-1. mRNA data for MAFbx in control and dexamethasone treated cells. Values are means SE. Transcripts are normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA and expressed relative to control value. Significantly different from *control and dexamethasone (within group); p<0.05. Treatment Dex (-) Dex (+) Control 1.0400.149 6.1220.408* L-Arg (10mM) 0.8070.000* 4.3210.426* DETA-NONO (10M) 0.7160.088* 3.9040.476* Table 4-2. mRNA data for CAT transporters in cell culture. Values are means SE. Transcripts are normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA and expressed relative to control value. Significantly different from *control; p<0.05. Gene of interest Control Glucocorticoid CAT-1 2.534 1.229 0.107 0.071* CAT-2 1.503 0.3945 0.242 0.129* Table 4-3. Body and muscle masses and total protein content of animals. Values are means SE. There was no value significantly different from control; body mass p=0.170, gastrocnemius mass p=0.490, total protein/ muscle p=0.087. Control Glucocorticoid Body Mass (g) 36.4 0.95 34.4 1.18 Gastrocnemius Mass (g) 150.2 5.75 154.8 3.96 Total Protein (mg)/ Muscle (g) 15.81 0.74 13.61 0.90 Table 4-4. mRNA data for CAT transporters in animals. Values are means SE. Transcripts are normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA and expressed relative to control value. No value was significantly different from control (CAT-1 p=0.221; CAT-2 p=0.18). Gene of interest Control Glucocorticoid CAT-1 1.740 0.699 3.146 0.820 CAT-2 1.115 0.281 2.002 0.548 39

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240kDa 150kDa 46kDa A) B) 00.0010.0020.0030.0040.005CDextreatmentscleaved/ total alpha-spectrin (optical density) Figure 4-1. Protein analysis -spectrin A) representative immunoblot for -spectrin (total 240kDa; cleaved 150kDa) and -actin (loading control; 46kDa) in L6 myotubes treated with 24h dexamethasone or vehicle. Lanes 1 4 are control, lanes 5 8 are Dexamethasone (Dex). B) quantification of cleaved-to-total -spectrin ratio. No value was statistically significant (p=0.818). 40

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Figure 4-2. Representative images from cells fixed in 3.9% formaldehyde and H & E stained. A) control B) dexamethasone (Dex) C) 10mM L-Arg D) Dex + 10mM L-Arg E) 10M DETA-NONO F) Dex + 10M DETA-NONO. 41

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01000200030004000500060007000CArgNOTreatmentsmyotube area Control Dex Figure 4-3. Average area of individual myotubes. Quantification of myotube area. Values are means SE. Significantly different from *control and dexamethasone; p<0.05. 051015202530CArgNOTreatmentsnuclear number/ myotube Control Dex Figure 4-4. Nuclear number per myotube. Quantification of nuclear number. Values are means SE. Significantly different from *control and dexamethasone; p<0.05. 42

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A) nNOS eNOS B) 0.0000.2000.4000.6000.8001.0001.200CONGCGroupArbitrary Unit s 0.0000.2000.4000.6000.8001.0001.200CONGCGroupArbitrary Units nNOS eNOS Figure 4-5. Protein analysis nNOS and eNOS A) representative immunoblot for nNOS and eNOS in mouse muscle homogenate treated with 8wk prednisolone or vehicle. B) quantification of nNOS and eNOS normalized to control. 43

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0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 0 M 5 M 10 M50 MDETA-NO Concentration Percent of Fibers with Emanating MyoD+ Control GC Figure 4-6. Satellite cells emanating from activated fibers. Quantification of cells emanating from activated fibers. Supplementation of culture media with DETA-NONO (5-50 M) induced satellite cell emanation in a dose-dependent manner. 44

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CHAPTER 5 DISCUSSION Glucocorticoids cause substantial skeletal muscle atrophy (34, 77) and suppress NO production (42, 81). We show that 24h of treatment with glucocorticoids in cell culture increases MAFbx mRNA expression and that an NO donor attenuates the increased expression. After 48h of glucocorticoid treatment, we witness a dramatic decrease in myotube dimensions, which is partially rescued by either L-Arg or nitric oxide donation. We failed to confirm our hypothesis that NO production is compromised in glucocorticoid treated cells, as there was no change in nitrate accumulation in 24h media collection. In addition to these acute effects observed with glucocorticoid use, we demonstrate that eight weeks of corticosteroid treatment reduces satellite cell activation/ proliferation from isolated fibers, which is necessary for skeletal muscle remodeling. Moreover, nNOS and eNOS protein content are reduced in glucocorticoid treated gastrocnemius. Interestingly, we found NO donation restores satellite cell emanation from glucocorticoid treated fibers to levels of control fibers, suggesting NO availability may be partially responsible for glucocorticoid-induced myopathy, in vivo. Acute Effects of Glucocorticoids Myotube Dimensions Glucocorticoids induce protein loss and a decrease in myotube size (23). We show that 48h dexamethasone treatment reduces myotube dimensions a decrease in size of 64%. Additionally, dexamethasone treatment resulted in less coverage of the field of view by myotubes and in fewer nuclei per myotube. We hypothesized that the atrophic myotubes were a result of reduced NO availability. 45

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Initially to test this, we supplemented cultures with either L-Arg (10mM) or DETA-NONO (10M). With these additions, the reduction in myotube size associated with dexamethasone treatment was attenuated by 22%. Furthermore, supplement inclusion resulted in maintenance of nuclear number. These results suggest, at least in part, that glucocorticoid-induced atrophy can be rescued with nitric oxide. NO Availability While the histological morphology data imply that NO availability may be compromised, these measurements fail to specifically demonstrate a reduction in NO production by glucocorticoid treated cells. In order to test this hypothesis we indirectly measured NO production by assessing nitrate and nitrite accumulation in the media. Fluorescence was constant in all samples. As we were unable to measure changes in nitric oxide production, we cannot confirm a limitation in the skeletal muscles ability to produce nitric oxide. And, even though our attempt to verify NOS protein in L6 lysate was inconclusive, we are confident these cells possess this protein (10), and thus are capable of synthesizing nitric oxide. Calpain Activation Glucocorticoid administration likely induces skeletal muscle atrophy through increased expression of genes involved in proteolysis (17, 19, 38, 106). The inhibition of calcium-activated proteases (calpains) in glucocorticoid treated myotubes attenuates the usual glucocorticoid-induced protein loss and decrease in myotube size (77). And while it is generally accepted that calpain activation is a key initial event in protein degradation, we failed to show this in atrophy associated with glucocorticoid use. We showed atrophy in glucocorticoid treated myotubes versus controls can occur without a concomitant increased in cleaved versus total -spectrin, a structural protein targeted by calpains. This is in agreement with Banik et al. (4), who 46

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found a methylprednisolone-induced dose-dependent inhibition of rabbit muscle calpain activity. They also demonstrated enzyme inhibition with dexamethasone and prednisolone treatments. Another group (25) found a decrease in calpain activity and -spectrin cleavage in mechanically ventilated rat diaphragm following glucocorticoid treatment. Additionally, Wang et al. (106) demonstrated only a small contribution of calcium-dependent muscle proteolysis in L6 myotube dexamethasone-induced atrophy. Furthermore, groups showing a positive correlation between dexamethasone treatment and calpain activity were actually measuring enzymatic activity. We analyzed the result of calpain activity in the living cell by measuring cleaved versus total -spectrin protein, as the measurement of in vitro maximal enzymatic activity may not represent in vivo proteolytic activity. Further measurements will be necessary to draw firm conclusions regarding the role of calcium-dependent muscle proteolysis in glucocorticoid-induced myopathy. MAFbx Control MAFbx has at least three controls. It is regulated by two elements of the IGF-1/ Akt pathway and by one component of the atrophy pathway (Figure 5-1). Akt has two routes for diminishing MAFbx expression. It blocks FOXO, which is directly responsible for upregulation of MAFbx and enhances mTOR activity, which not only increases p70S6K, but also blocks MAFbx (26, 50). In addition to these events in the hypertrophy pathway that inhibit MAFbx expression, it has been shown that p38 via TNF signaling in the atrophy pathway stimulates MAFbx expression. We have demonstrated that 24h of treatment with glucocorticoids in cell culture increases MAFbx mRNA expression and that an NO donor attenuates the increased expression. It is not totally clear why an upregulation of MAFbx is seen with glucocorticoid use. IGF-1 and insulin both stimulate the hypertrophy pathway (Figure 5-1), and insulin resistance develops with 47

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glucocorticoid use (70). Also it has been shown that NOS is essential for skeletal muscle hypertrophy (84). So, perhaps instead of solely increasing activity of the atrophy pathway, the hypertrophic signals are attenuated, thus removing the blockade on FOXO and MAFbx via deactivated Akt, with the ultimate effect of dramatic skeletal atrophy. Nitric oxides attenuation of the glucocorticoid effect on MAFbx mRNA is likely though the hypertrophy pathway, as it has been shown that NOS is essential for skeletal muscle hypertrophy (84). Possible Mechanisms of Action for Nitric Oxide Nitric oxide has been shown to be a key regulator of muscle hypertrophy and satellite cell activation. It has not been completely elucidated how nitric oxide is having its effect, but perhaps it is through some component of the hypertrophy pathway. This would explain its ability to attenuate glucocorticoid-induced atrophy. We have shown that adequate NO availability may be a limitation in skeletal muscle myopathy following glucocorticoid treatment. The fact that we observed dramatic recovery of myotube dimensions and nuclear number and partial attenuation of MAFbx mRNA leads us to believe NO is potentially acting on a downstream target of the hypertrophy (IGF-1) pathway. For example, in arteries with functional endothelial cells, IGF-1 caused a concentration-dependent relaxation (78). This effect was abolished by the use of a NOS inhibitor. Futhermore, this author demonstrated formation of NO in response to IGF-1 treatment in cultured endothelial cells, and that the signaling was independent of intracellular Ca 2+ but involved PI3K, a direct downstream target of IGF-1. PI3K is required to phosphorylate (activate) Akt. Perhaps glucocorticoids attenuate Akt phosphorylation, which would lower activation of mTOR and activate FOXO thereby leading to atrophic signaling through MAFbx. Supplementation with exogenous L-arginine or nitric oxide reverses the atrophy by suppressing MAFbx activation. 48

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Furthermore, MAFbx may be directly upregulated with glucocorticoid use due to FOXO activation. GSK3 is blocked by phosphorylated (active) Akt, and leads to hypertrophy (73) and FOXO inactivation (Figure 5-2). Sandri et al. (77) has shown that this same pathway, when inhibited at the IGF-1 level, will increase FOXO activation. We show the upregulation of MAFbx seen with glucocorticoid treatment was moderately attenuated by use of L-arginine, a NOS substrate, and NO donation (via DETA-NONO). Thus, the mild attenuation of MAFbx upregulation seen with the treatment of NO or L-Arg could be a result of any one of multiple elements in the IGF-1 pathway. Due to the lack of apparent calpain activation, which seems to not involve PI3K, it would be more reasonable to think that NO is working at least at the Akt level, if not further downstream. Further studies are necessary to determine if Akt and or FOXO are directly involved in NO action in skeletal muscle maintenance. CAT Expression in Cultured Myotubes The reduced expression of the CAT transporters seen in cell culture agrees with Simmons et al. (81) who showed a reduction in L-Arg uptake in dexamethasone treated cells. Dexamethasone treatment prevented the induction of CAT-2B mRNA by cytokines. In further agreement with our findings, Hammermann et al. (33) found a down-regulation of CAT-2B in rat alveolar macrophages after 20h exposure to dexamethasone. It seems that the inflammatory response increases L-Arg transport into the cells, which supports large-scale NO production by iNOS. Dexamethasone, however, is able to not only attenuate this increase in L-Arg influx by the down-regulation of CAT transporters, but due to its anti-inflammatory characteristics also seems to suppress iNOS (82). Our evidence supports this in that CAT-2 mRNA expression was down-regulated with dexamethasone treatment, however we were unable to measure changes in NOS. Therefore, glucocorticoids have the ability to reduce NO production by inhibiting 49

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substrate availability through lower arginine transport into the cell. Given the importance of NO signaling for skeletal muscle remodeling as discussed above, inhibition of endogenous NO production may be an important mechanism for glucocorticoid-induced skeletal muscle myopathy. Futhermore, if the mechanism to synthesize NO is compromised with glucocorticoid treatment, then an NO donor should attenuate the effects of the glucocorticoid better than substrates for NO synthesis. This phenomenon may explain how DETA-NONO was a superior to L-Arg for inhibiting MAFbx expression. L-Arg only decreased the glucocorticoid-induced MAFbx mRNA expression by 1.8 fold, while DETA-NONO had a marginally better effect of a 2.3 fold decrease in expression. Without as much NOS or CAT present, physiological levels of L-Arg might not have been sufficient. But, bypassing the NOS enzyme, as was done using the NO donor DETA-NONO, a greater effect was observed. Chronic Effects of Glucocorticoids NOS Expression in Mouse Skeletal Muscle After 8 weeks of prednisilone administration, we report a drop in both eNOS and nNOS expression in mouse skeletal muscle. This suggests that corticosteroids actually lead to lower amounts of the enzyme, which could result in reduced nitric oxide production and possible changes in both hypertrophic and atrophic signaling pathways. eNOS down-regulation seems to be the cause of the lack of NO production observed with glucocorticoid treatment in cardiac microvascular endothelial cells (81). eNOS has been shown to be down-regulated by FOXO1 and FOXO3A (69). These factors bind to the eNOS promoter in endothelial cells. This causes repression of eNOS expression, and subsequent NO generation. So, the acute effects of glucocorticoids may be upregulation of MAFbx expression via FOXO action, with FOXO being the culprit of the long-term effect of decreased NOS expression. 50

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CAT Expression in Mouse Skeletal Muscle NOS presence is not the sole factor in determining NO production. L-Arg, a substrate for NOS, must be transported into the cell via the CAT transporters. These transporters had greater mRNA expression in the animals. Perhaps the increase in expression seen in the animals was due to a more chronic exposure to the corticosteroid. As NOS expression continued to decline over the 8 weeks, an upregulation in the cationic transporter was possibly an attempt to counter the decrease in eNOS and nNOS and restore NO levels. Since we did not directly measure NO production, we cannot confirm the net effect of decreased NOS expression and increased CAT transporter expression. Nevertheless, the efficacy of NO supplementation to isolated myofibers implies that endogenous NO production may have been compromised in the GC group. Satellite Cell Activity in Single Muscle Fibers In addition to NO inhibiting the synthesis of MAFbx and thus diminishing the initial atrophy effect, it can also be used to rescue satellite cells from glucocorticoid treated animals. It has been shown by numerous labs that activation of satellite cells is a necessary step in adult skeletal muscle growth and regeneration (1, 61, 67, 74). Futhermore, our work confirms these cells may be critical for maintenance of muscle mass. Other labs have demonstrated this in atrophied muscles from hindlimb suspended animals. Isolated myofibers from these animals contained fewer satellite cells and these cells had an impaired ability to activate and proliferate (62). This de-activation may be yet another factor in atrophy. In our study, we found a significant reduction in satellite cells emanating from glucocorticoid-treated fibers in response to mechanical stimulation. Approximately half of the fibers from control animals had at least one emanating myoD+ mononuclear cell, while only one-fourth of the fibers from glucocorticoid treated animals had emanating satellite cells. 51

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Not only did we confirm that satellite cell function is compromised with chronic glucocorticoid treatment, but we also show exogenous NO treatment can ameliorate the glucocorticoid-induced deficit in satellite cell activation/ proliferation. In other models this has been a successful treatment for satellite cell activation (2, 3, 94, 95). Our findings suggest that NO production may be reduced with prolonged glucocorticoid exposure, and this is what reduces satellite cell activity. 52

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Atrophy Pathway Hypertrophy Pathway Figure 5-1. Illustration of the muscle maintenance pathways insulin IGF-1 PI3K Akt GSK3 TSC1/2 mTOR eIF2B p 70S6 K MAFbx FOXO Inflammatory C y tokines p 38 IK K N F K B MURF1 L-Arg Satellite Cell Activation nNOS eNOS Calpain N O MAFbx Cleaves actin Caspase-3 L-Arg CAT Figure 5-2. Illustration of potential effects of nitric oxide. 53

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CHAPTER 6 CONCLUSION Glucocorticoid treatment both in vitro and in vivo has detrimental effects. Acutely, we confirmed they cause upregulation of a key element of the Ub-prot pathway (MAFbx). Chronic treatment decreases constitutive NOS, thus potentially compromising NO production. NO has been shown to be expressed in conjunction with activation of the IGF-1 pathway and critical to satellite cell activity, both of which play a role in muscle mass maintenance. We demonstrate that NO attenuates the expression of MAFbx with acute glucocorticoid treatment. Furthermore we show that NO can rescue satellite cell activity from muscle chronically treated with glucocorticoids. So, NO may impact both the atrophic and hypertrophic pathways in its role in muscle mass maintenance. NO supplementation to cultures did have positive effects on maintaining myotube dimensions and increasing satellite cell activity. So, even if it does not directly inhibit a component of the atrophy pathway, NO may be a viable treatment option by augmenting muscle mass maintenance from the hypertrophy side via satellite cell activity and fusion with the existing myotubes. Thus, enhancing NO production may be important in attenuating the atrophic effects associated with both acute and chronic glucocorticoid use. 54

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BIOGRAPHICAL SKETCH Jodi Heather Dixon Long was born in DuPage County, Illinois. The older of two children she grew up mostly in Carol Stream, Illinois, but moved with her family to St. Simons Island, Georgia in 1985 where she graduated salutatorian of the class of 1989 from Frederica Academy. In 1993, she earned her B.A. in mathematics, with a minor in secondary education from Erskine College in Due West, S.C. Upon baccalaureate graduation, Jodi served as a middle and high school math and science teacher and taught group exercise. In 1997, Jodi returned to school to earn a M.S. in exercise physiology from Georgia Southern University. She was a graduate assistant in the physical education program while earning this degree. Following graduation in December of 1998, Jodi returned to a teaching career. She taught physical activity classes and anatomy and physiology at Coastal Georgia Community College in Brunswick, GA. In the fall of 1999, Jodi moved to a full time instructor position at her alma mater of Georgia Southern University where she taught anatomy and physiology, exercise physiology and health survey courses. Once again, Jodi returned to school to complete her terminal degree. In the fall of 2002, she began studies at the University of Florida under the direction of David S. Criswell. While studying at UF, Jodi also taught anatomy and physiology at Santa Fe Community College in Gainesville, FL, eventually becoming a full time faculty member. Following graduation from the doctor of philosophy program in the College of Health and Human Performance (Department of Applied Physiology and Kinesiology), Jodi plans to continue to educate college students in anatomy and physiology at Santa Fe Community College. She also continues to teach group fitness. 66

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In addition to her professional and academic achievements, Jodi was married in 1991 to George W. Trey Long, III of McCormick, SC and they have two children, Sam (6yr) and Alex (4yr). 67