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Reduced Sensitivity to Nitric Oxide Limits Satellite Cell Activity on Cultured Myofibers from Aged Mice

Permanent Link: http://ufdc.ufl.edu/UFE0021194/00001

Material Information

Title: Reduced Sensitivity to Nitric Oxide Limits Satellite Cell Activity on Cultured Myofibers from Aged Mice
Physical Description: 1 online resource (72 p.)
Language: english
Creator: Betters, Jenna Leigh Jones
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: 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: Although aging-induced deficits in satellite cell proliferation and muscle regeneration are well-characterized, our investigation provides novel evidence of an age-related deficit in satellite cell activation, independent of proliferation, that is intrinsic to the muscle fiber. Mechanical activation of muscle fibers induces enzymatic production of nitric oxide (NO) resulting in proteolytic release of hepatocyte growth factor (HGF) from its extracellular tethering, and subsequent activation of satellite cells. We hypothesized that aging would reduce in situ satellite cell activation, and that L-arginine or nitric oxide (DETA-NO) supplementation would improve satellite cell activity in old muscle. Single intact myofibers were isolated from the gastrocnemius muscles of young (2 mo), adult (10 mo), and aged (22 mo) male C57 mice. Fibers were centrifuged to stimulate satellite cell activation and incubated in serum-free media with 0.002% 5-bromodeoxyuridine (BrdU) and either 2 mM L-arginine, 10 ?M DETA-NO, or control media for 48 h. The total number of satellite cells per fiber (i.e., pax7+) was unchanged with aging. Nevertheless, the number of activated satellite cells (BrdU+) following centrifugation was reduced in the aged fibers. L-arginine or DETA-NO treatment to the aged fibers restored satellite cell activation to young-control levels. However, these treatments also increased satellite cell activation in young and adult fibers, such that an age-dependent deficit in satellite cell activity persisted within treatment groups. Expression of neuronal and endothelial nitric oxide synthase (NOS) proteins in the plantaris muscles did not differ between age groups. Further, contraction-induced NO release from the extensor digitorum longus (EDL) did not differ between adult and old mice. These data suggest that impairment of NO production does not account for reduced satellite cell activity in aged muscle fibers. Also, treatment with exogenous HGF was equally effective in activating satellite cells across age groups, indicating that events downstream of HGF release are intact in the aged muscle. Therefore, these data imply a reduced sensitivity of aged muscle to NO. Insight gained from these experiments will augment therapeutic strategies to maintain skeletal muscle mass in the elderly, and to enhance muscle regeneration in this population following disuse or acute injury.
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 Jenna Leigh Jones Betters.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Criswell, David S.

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Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021194:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021194/00001

Material Information

Title: Reduced Sensitivity to Nitric Oxide Limits Satellite Cell Activity on Cultured Myofibers from Aged Mice
Physical Description: 1 online resource (72 p.)
Language: english
Creator: Betters, Jenna Leigh Jones
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: 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: Although aging-induced deficits in satellite cell proliferation and muscle regeneration are well-characterized, our investigation provides novel evidence of an age-related deficit in satellite cell activation, independent of proliferation, that is intrinsic to the muscle fiber. Mechanical activation of muscle fibers induces enzymatic production of nitric oxide (NO) resulting in proteolytic release of hepatocyte growth factor (HGF) from its extracellular tethering, and subsequent activation of satellite cells. We hypothesized that aging would reduce in situ satellite cell activation, and that L-arginine or nitric oxide (DETA-NO) supplementation would improve satellite cell activity in old muscle. Single intact myofibers were isolated from the gastrocnemius muscles of young (2 mo), adult (10 mo), and aged (22 mo) male C57 mice. Fibers were centrifuged to stimulate satellite cell activation and incubated in serum-free media with 0.002% 5-bromodeoxyuridine (BrdU) and either 2 mM L-arginine, 10 ?M DETA-NO, or control media for 48 h. The total number of satellite cells per fiber (i.e., pax7+) was unchanged with aging. Nevertheless, the number of activated satellite cells (BrdU+) following centrifugation was reduced in the aged fibers. L-arginine or DETA-NO treatment to the aged fibers restored satellite cell activation to young-control levels. However, these treatments also increased satellite cell activation in young and adult fibers, such that an age-dependent deficit in satellite cell activity persisted within treatment groups. Expression of neuronal and endothelial nitric oxide synthase (NOS) proteins in the plantaris muscles did not differ between age groups. Further, contraction-induced NO release from the extensor digitorum longus (EDL) did not differ between adult and old mice. These data suggest that impairment of NO production does not account for reduced satellite cell activity in aged muscle fibers. Also, treatment with exogenous HGF was equally effective in activating satellite cells across age groups, indicating that events downstream of HGF release are intact in the aged muscle. Therefore, these data imply a reduced sensitivity of aged muscle to NO. Insight gained from these experiments will augment therapeutic strategies to maintain skeletal muscle mass in the elderly, and to enhance muscle regeneration in this population following disuse or acute injury.
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 Jenna Leigh Jones Betters.
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: UFE0021194:00001


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REDUCED SENSITIVITY TO NITRIC OXIDE LIMITS SATELLITE CELL ACTIVITY ON
CULTURED MYOFIBERS FROM AGED MICE




















By

JENNA LEIGH JONES BETTERS


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 Jenna Leigh Jones Betters


































To my husband, Chad Betters, who never doubted my capabilities, even when I did









ACKNOWLEDGMENTS

This work was completed with the help of many people. First, I thank Dr. David Criswell,

my supervisory committee chair. I gained valuable knowledge during my 6 years working in his

lab. He guided me through the completion of this often-daunting degree.

I thank my committee members (Drs. Scott Powers and Steve Dodd from Applied

Physiology and Kinesiology, and Dr. Sally Johnson from Animal Sciences). Each has

contributed valuable insight to this project, and to my graduate experience.

Dr. Grace Pavlath and Brenda Bondesen from Emory University graciously taught me the

fiber isolation technique. I greatly appreciate their expertise and willingness to assist me.

The members of the Molecular Physiology lab deserve some credit, too. Quinlyn Soltow,

Vitor Lira, and Jeff Sellman, have been my fellow "lab rats" throughout my graduate student

tenure. Vitor and Dana Brown deserve extra credit for all the time spent "on the Dark Side" to

complete the DAF experiments.

Lastly, I would not be in graduate school and completing this dissertation without the love

and support of my family. My husband, Chad Betters, and my parents and sister, Jim and Sue

Jones and Julie Jones, provided the "behind the scenes" support that made this work possible.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S ................. ...............7....__.......


LI ST OF FIGURE S .............. ...............8.....


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


CHAPTER


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


Background ................. ...............12.................
Problem Statement ................. ...............13.................
Variables in Study............... ...............14.
Specific Aims and Hypotheses ................ ...............14................
Limitations/Delimitations/Assumptions ................. ...............16.................
Significance of the Study ................. ...............16................

2 LITERATURE REVIEW ................. ...............17................


Satellite Cell Activation....................... ...........1

Hepatocyte Growth Factor (HGF) ................. ...............18................
Nitric Oxide (NO)............... ...............18..
Stretch .................. ........... ...............20.......
Recovery from Atrophy ................... .......... ...............21......
Aging and Muscle Satellite Cell Activity ................. ...............23...............
Satellite Cell Numbers with Age ................. ...............23........... ...
Aging and Muscle Regeneration ................ ...............25...
Notch Signaling and Satellite Cell Activation............... ...............2
Nitric Oxide and HGF/c-Met in Aged Muscle ......____ ..... ... .__ ........_.......3
Sum mary ............ ..... ._ ............... 1....

3 MATERIALS AND METHODS .............. ...............33....


Experimental Designs ............ ..... ._ ...............33....
A nim als............... .... ....... ... ...... ..... ...............3
In Vitro Electrical Stimulation and Measurement of Nitric Oxide Production ......................34
Fiber Isolation ...._ .. ................. ......._ .. ..........3
Immunohistochemistry ........................... ...... ... ........3
Asymmetric Dimethylarginine (ADMA) ELISA ................. ...............38................
W western Blotting ................. ...............38.................
Statistical Analysis............... ...............39











4 RE SULT S .............. ...............40....


Muscle Atrophy in Male C57 Mice .........__..... ......__. ...............40.
Nitric Oxide Production from Contracting Muscle .............. ...............40....
Nitric Oxide Synthase Protein Expression Is Not Reduced............... ...............41
Asymmetric Dimethylarginine Can Inhibit Satellite Cell Activity .............. ....................41
Asymmetric Dimethylarginine Is Not Elevated in Aged Muscle and Blood .........................42
Satellite Cell Response to HGF Is Not Altered with Age ................ ... ....... ....... ..........4
L-Arginine and a Nitric Oxide Donor Can Rescue Aged Satellite Cell Activity .................42

5 DI SCUS SSION ............ ..... ._ ...............56...


M ain Findings ................ ... .... .._ ... ...._ .......... .................5
Reduced Satellite Cell Activation in Response to Mechanical Perturbation..........................56
L-Arginine and a Nitric Oxide Donor Can Rescue Aged Satellite Cell Activity .................57
Nitric Oxide Production and NOS Protein Expression from Aged Muscle ...........................58
Satellite Cell Response to HGF Is Not Altered with Age .............. .. ..... .. ....... ... .........6
Asymmetric Dimethylarginine Is Not Elevated in Aged Muscle and Blood but Does
Inhibit Satellite Cell Activity .............. ...............60....
Response to Nitric Oxide ............ ...... ..__ ...............62..
Limitations and Future Directions ............_ ..... ..__ ...............63..
Conclusions............... ..............6

LIST OF REFERENCES ............_ ..... ..__ ...............67...

BIOGRAPHICAL SKETCH .............. ...............72....










LIST OF TABLES


Table page

4-1 Body mass, gastrocnemius mass, muscle mass/body mass ratios, and total protein for
young, adult, and old mice. .............. ...............44....

4-2 Asymmetric dimethylarginine (ADMA) concentration (Cpmol/L) in serum and muscle
of young and old mice. ................. ...............45._..._.....

4-3 Quantification of satellite cell activation as measured by (A) cell emanation and (B)
BrdU incorporation from young, adult, and old myofibers. ............. .....................4










LIST OF FIGURES


Figure page

4-1 Nitric oxide (NO) production as quantified by 4,5-diaminofluorescein (DAF)
fluorescence. ............. ...............47.....

4-2 Representative immunoblot of neuronal nitric oxide synthase from young, adult, and
old plantaris muscle. ............. ...............48.....

4-3 Representative immunoblot of endothelial nitric oxide synthase from young, adult,
and old plantaris muscle............... ...............49.

4-4 Representative images of an emanating satellite cell and a BrdU' nucleus. .....................50

4-5 Satellite cell emanation and BrdU incorporation of fibers following exposure to
ADM A ............. ...............51.....

4-6 Representative image of a fiber stained for pax7 immediately following isolation and
plating. ............ ...............52.....

4-7 Satellite cell emanation and BrdU incorporation of fibers following exposure to
HGF ........... _.......__ ...............53....

4-8 Satellite cell emanation and BrdU incorporation of fibers following exposure to L-
arginine and DETA-NO. ............ ...............54.....

5-1 Overview of the steps leading to satellite cell activation and the potential location of
the aging def cit ................. ...............66........... ....









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

REDUCED SENSITIVITY TO NITRIC OXIDE LIMITS SATELLITE CELL ACTIVITY ON
CULTURED MYOFIBERS FROM AGED MICE

By

Jenna Leigh Jones Betters

August 2007

Chair: David Criswell
Maj or: Health and Human Performance

Although aging-induced deficits in satellite cell proliferation and muscle regeneration are

well-characterized, our investigation provides novel evidence of an age-related deficit in satellite

cell activation, independent of proliferation, that is intrinsic to the muscle fiber. Mechanical

activation of muscle fibers induces enzymatic production of nitric oxide (NO) resulting in

proteolytic release of hepatocyte growth factor (HGF) from its extracellular tethering, and

subsequent activation of satellite cells. We hypothesized that aging would reduce in situ satellite

cell activation, and that L-arginine or nitric oxide (DETA-NO) supplementation would improve

satellite cell activity in old muscle. Single intact myofibers were isolated from the gastrocnemius

muscles of young (2 mo), adult (10 mo), and aged (22 mo) male C57 mice. Fibers were

centrifuged to stimulate satellite cell activation and incubated in serum-free media with 0.002%

5-bromodeoxyuridine (BrdU) and either 2 mM L-arginine, 10 CLM DETA-NO, or control media

for 48 h. The total number of satellite cells per fiber (i.e., pax7 ) was unchanged with aging.

Nevertheless, the number of activated satellite cells (BrdU ) following centrifugation was

reduced in the aged fibers. L-arginine or DETA-NO treatment to the aged fibers restored satellite

cell activation to young-control levels. However, these treatments also increased satellite cell

activation in young and adult fibers, such that an age-dependent deficit in satellite cell activity










persisted within treatment groups. Expression of neuronal and endothelial nitric oxide synthase

(NOS) proteins in the plantaris muscles did not differ between age groups. Further, contraction-

induced NO release from the extensor digitorum longus (EDL) did not differ between adult and

old mice. These data suggest that impairment of NO production does not account for reduced

satellite cell activity in aged muscle fibers. Also, treatment with exogenous HGF was equally

effective in activating satellite cells across age groups, indicating that events downstream of

HGF release are intact in the aged muscle. Therefore, these data imply a reduced sensitivity of

aged muscle to NO. Insight gained from these experiments will augment therapeutic strategies to

maintain skeletal muscle mass in the elderly, and to enhance muscle regeneration in this

population following disuse or acute injury.









CHAPTER 1
INTTRODUCTION

Sarcopenia, the gradual decline of skeletal muscle mass and strength, is a hallmark of

aging. Sarcopenia is generally responsible for the age-related decline in physical performance,

and ultimately for physical disability in many elderly people. The prevalence of sarcopenia,

defined as appendicular skeletal muscle mass (kg)/height2 (m2) being less than two standard

deviations below the mean of a young reference group, ranges from 13 to 24% in those 65 to 70

years old, and is over 50% for those greater than 80 years of age (6). Another associated

characteristic of aging is a decline in ability of skeletal muscle to repair itself following damage.

Age-associated muscle atrophy likely results, in part, from the reduced ability of muscle to

regenerate following repeated micro-trauma occurring during the course of daily activities (e.g.,

eccentric contraction-induced injury).

Mature skeletal muscle fibers consist of post-mitotic and terminally differentiated nuclei.

Thus, the addition of nuclei to skeletal muscle fibers during hypertrophy or regeneration must

come from sources other than myonuclei. Satellite cells represent the muscle "stem" cells that

provide these additional nuclei. Satellite cells are quiescent precursor cells residing in a

"satellite" position alongside muscle fibers (37). The ability of these muscle precursor cells to

increase muscle mass and repair muscle following injury makes them essential to skeletal muscle

function. However, with advanced age, there is a reduction in the population of satellite cells (5,

12, 28, 31, 45, 48, 51) and their ability to activate/proliferate, and repair muscle (19, 20). Thus,

the ability of aged muscle to increase mass or repair damage is poor compared to young or adult

muscle. The mechanisms underlying the age-related deficit in satellite cell function warrant

exploration.









Background

Muscle atrophy during aging likely results, in part, from inefficient muscle repair

consequent to injury. Skeletal muscle regeneration following immobilization-induced atrophy is

lacking in aged rats, even after 77 days of recovery (17). However, direct administration of IGF-

1 onto atrophied muscles restores the in vivo regenerative capacity of old muscle and in vitro

satellite cell proliferation. This finding suggests that satellite cells from aged muscle do not lose

their inherent ability to proliferate, but simply are not activated sufficiently to regenerate

following an injury. Thus, it is important to isolate the process of activation from subsequent

proliferation. Old satellite cells may remain inactive due to the lack of some unknown growth

factor/mitogen, or the failure to respond to the levels of endogenous growth factors.

Satellite cells are known to be activated by several stimuli, such as hepatocyte growth

factor (HGF) (4, 53, 54), mechanical stretch (54, 59) and exercise (23, 24, 32). Activation of

satellite cells refers to entry into the cell cycle from quiescence, including DNA synthesis, and

mobilization. Other growth factors, such as IGF-1 and basic FGF, can stimulate proliferation of

satellite cells (7, 8, 9, 10, 51, 61) but thus far, only HGF affects activation.

Nitric oxide (NO) appears to be an important signaling molecule mediating satellite cell

activation via HGF release (4). Inhibition of NO production inhibits HGF release, c-met/HGF

co-localization, and satellite cell activation (2). In addition, mechanical stretch of rat satellite

cells (54) and isolated mouse myofibers (59) identified the importance of NO in stretch-induced

satellite cell activation. Similarly, HGF is crucial for stretch-induced satellite cell activation, and

its release is dependent on NO (54).

Satellite cell proliferation and cell fate determination are regulated by the Notch signaling

pathway. This pathway is triggered by the rapid upregulation of Delta, the Notch ligand,

following injury. Recently, Conboy et al. (19) demonstrated deficiencies in Notch signaling that









affected proliferation and regeneration of old mouse muscle. They showed that old muscle fails

to upregulate Delta in response to an injury. Forced activation of Notch improved regeneration in

old muscle, while Notch inhibition decreased repair in young muscle. When old cultured satellite

cells were exposed to young serum, satellite cells upregulated Delta and activated Notch (20). In

addition, parabiotic pairing of a young and an old mouse, such that they shared a circulatory

system, restored regenerative potential following an injury in the old mouse muscle. These

experiments demonstrate that Notch activation is necessary for regeneration in young muscle and

sufficient to stimulate effective repair in old muscle. Plus, components in young serum are

capable of reversing the age-induced decline in activation/proliferation of satellite cells. Thus,

satellite cells do not lose their innate ability to repair muscle when aged, but modifications of the

old systemic environment prevent their proper functioning.

Problem Statement

A better understanding of sarcopenia is essential since a significant proportion of the

population is living long enough that muscle atrophy becomes a maj or issue. In addition,

physical rehabilitation following an injury requires functional satellite cells. Aging is associated

with an impaired ability to repair muscle following damage, and an increased amount of muscle

wasting. Advanced age reduces satellite cell numbers (5, 12, 28, 31, 45, 48, 51) and satellite cell

activation/proliferation (19, 20). Work by Conboy and colleagues (20) has shown that aged

satellite cells can repair muscle when exposed to a young systemic environment. This suggests

that extrinsic signals regulate aged satellite cell activity, and that the possibility exists that the

loss of muscle regenerative capacity can be restored if the satellite cell environment more closely

resembles that of a young animal. Given the importance of NO as the initiating signal for

satellite cell activation, we propose that one key age-related modification in the muscle

environment is a decrease in NO bioavailability. We postulate that NO production is reduced









with aging and may compromise satellite cell function. NO production could be reduced due to a

decrease in the cationic amino acid transporter (CAT) expression responsible for transporting the

NOS substrate L-arginine into the cell, a reduction in NOS expression, or an increase in

asymmetric dimethylarginine (ADMA), an endogenously produced metabolite which inhibits

NOS activity. Discovery of the age-associated factors that regulate satellite cell behavior will be

instrumental for designing therapies targeted to augment the regenerative potential of muscle and

thus maintain muscle mass in elderly individuals.

Variables in Study

Independent variables: We will manipulate the age of the animals, electrical stimulation of

incubated muscles, and the isolated myofibers' treatment medium (supplementing with L-

arginine, DETA-NO, HGF, or ADMA).

Dependent variables: We will measure satellite cell activation via BrdU incorporation. We will

also measure nNOS, eNOS, and iNOS protein content in plantaris muscle, CAT1/2 mRNA

levels, and stain for Pax7 and BrdU in isolated myofibers. We will measure nitric oxide

production in extensor digitorum longus (EDL) muscles electrically stimulated in vitro.

Control variables: Only male C57 mice will be studied, so gender is purposely excluded from

this study.

Extraneous variables: We will not control prior activity level or food and water intake of the

mice.

Specific Aims and Hypotheses

This study is designed to answer the following questions:

Question 1. Is nitric oxide production reduced in old muscle?










Hypothesis 1: NO production (quantified by DAF fluorescence) is reduced in old muscle

following in vitro electrical stimulation of the EDL. In addition, NOS protein expression and

CAT mRNA are reduced in old muscle.

Question 2. Is there a deficit in satellite cell activation with aging in response to exogenous

HGF?

Hypothesis 2. Aged satellite cells will respond to increasing doses of HGF by incorporating

BrdU into DNA similar to young satellite cells, indicating that the deficit is upstream of HGF

release.

Question 3. Is asymmetric dimethylarginine (ADMA) elevated in the muscle and/or blood of

aged animals?

Hypothesis 3: ADMA, as measured by a commercially available ELISA kit, is elevated in both

the serum and muscle of aged animals.

Question 4. Can ADMA inhibit satellite cell activation of young fibers?

Hypothesis 4: Physiologically relevant concentrations of ADMA will reduce satellite cell

activation of young fibers in response to mechanical perturbation (centrifugation).

Question Sa. Is there a deficit in satellite cell activation with aging in response to mechanical

stress?

Hypothesis Sa: Aged satellite cells will have decreased activation (cell emanation as well as

BrdU incorporation) of satellite cells in response to mechanical perturbation (centrifugation).

Question 5b. Can L-arginine and/or a nitric oxide donor (DETA-NO) rescue satellite cell

activation in old muscle?

Hypothesis 5b: Both L-arginine and DETA-NO treatment for 48 hours will return satellite cell

activation of old fibers to young levels.









Lim itations/D~elim itations/Ass umptions

Limitations: The invasive nature of this study negates the use of human subj ects. A mouse

hindlimb model has been selected because of the similarities in structure and function of mouse

and human skeletal muscle.

Delimitations: Gender and species differences may exist in regard to satellite cell function. We

have chosen to study only male C57 mice.

Assumptions: It is assumed that satellite cells remain quiescent during the myofiber isolation

procedure. Previous experiments have confirmed the quiescent nature of satellite cells during

isolation using this technique.

Significance of the Study

Normal daily activity subj ects skeletal muscle to injuries, necessitating a means of repair to

maintain muscle. Thus, there is a constant demand for satellite cells throughout life. A decrease

in satellite cell abundance and activity with age may contribute to the age-associated deficiency

in muscle repair, and ultimately to muscle loss.

This research will improve our knowledge of the mechanisms underlying the deficit in

satellite cell function with aging. We seek to identify aging-associated deficiencies in signaling

pathways leading to satellite cell activation, and to reverse these deficiencies. This study will

provide insight into clinical therapies to attenuate muscle loss and improve muscle regeneration

in the elderly.









CHAPTER 2
LITERATURE REVIEW

Skeletal muscle is a multi-nucleated tissue capable of generating force that is essential for

locomotion and breathing. It is continually subj ected to injury and micro-trauma, resulting from

mechanical stress, including weight bearing and exercise. Thus, skeletal muscle requires a

renewable source of cells for muscle repair. Satellite cells provide the additional nuclei to muscle

fibers in times of growth or regeneration.

Satellite cells are located adj acent to the muscle fiber and beneath the basal lamina (37).

They are believed to be the committed stem cells of adult skeletal muscle (57). While normally

quiescent, they are activated when needed for repair or to mediate muscle hypertrophy.

Aging is associated with a decreased satellite cell number (5, 12, 28, 31, 45, 48, 51) and

marked impairments in regeneration following injury or atrophy (17, 19, 20, 43). This project

seeks to understand the molecular mechanisms of satellite cell aging that lead to loss of tissue

regenerative capacity in old muscle.

Satellite Cell Activation

Satellite cell activation is defined as entry into G1 phase from quiescence, and mobilization

(2). To date, the only known factors to activate satellite cells include hepatocyte growth factor

(HGF) and nitric oxide (NO). Perturbations such as stretch and exercise act via NO and HGF

release to activate satellite cells. In addition, the activating factor in crushed muscle extract

(C1VE), a known activating treatment, is HGF (53). Other growth factors, such as insulin-like

growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF), cannot directly activate

satellite cells, but do improve proliferation (7, 8, 9, 10, 61).









Hepatocyte Growth Factor (HGF)

HGF is synthesized in an inactive form as a single chain of ~90 kDa, and is proteolytically

cleaved into an active, disulfide-linked heterodimer of 60 kDa (alpha-chain) and 30 kDa (beta-

chain) by serine proteases (40, 41). Tatsumi and Allen (52) showed that HGF is present in the

extracellular domain of uninjured rat skeletal muscle as active heterodimeric HGF. Significant

amounts of pro-HGF were only found in rat muscle when the integrity of the membrane was

compromised. Thus, active HGF is present in the extracellular matrix (ECM) and can be released

following injury. This is important because proteolytic conversion of pro-HGF to active HGF is

therefore not a rate-limiting step in the release of active HGF from the ECM.

HGF is released from its tethering to the ECM by the action of matrix metalloproteinases

(MMP's) (62). When released, HGF rapidly co-localizes with its receptor on satellite cells, the c-

met tyrosine kinase receptor, and triggers activation from quiescence (53). Once activated,

satellite cells synthesize both HGF and c-met (3, 53).

Treating muscle with CME causes dose-dependent activation of satellite cells (11, 53).

CME increases expression of proliferating cell nuclear antigen (PCNA), an indicator of

activation (DNA synthesis), in three week-old and nine month-old rat satellite cells (30). The

factor in CME causing activation was later identified as HGF (53). Treating satellite cells with

CME in the presence of anti-HGF neutralizing antibodies eliminated the CME-induced

proliferation, indicating that HGF is the factor in CME responsible for stimulating satellite cell

activity. Therefore, HGF is an important growth factor to skeletal muscle because of its role in

activating satellite cells and thereby contributing to repair and regeneration.

Nitric Oxide (NO)

Work has shown that HGF release from the extracellular matrix is NO-dependent, and

inhibition of nitric oxide synthase (NOS) prevents that release (2, 4, 50, 52). Anderson and









colleagues have demonstrated that NO synthesis is implicated in the activation of satellite cells in

crush injured skeletal muscle (2) and resident satellite cells on isolated myofibers cultured in

vitro (4). hz vivo, NOS inhibition prevents HGF/c-met colocalization and satellite cell activation

(2). Activation of satellite cells on single myofibers was found to be dependent on the levels of

NO (related to the amount of substrate, L-arginine), and HGF in the media (4). In contrast, NOS

inhibition (L-NAME) prevented this activation.

Anderson and Pilipowicz (4) characterized the series of events leading to satellite cell

activation on isolated single myofibers. CME, HGF, and L-arginine activated satellite cells,

while L-NAME significantly blocked the potent activation by CME, as well as spontaneous

control activation. These results suggest that NO plays a role in spontaneous and HGF-dependent

activation. Since spontaneous activation was decreased by NOS inhibition but restored by HGF,

the activation signaling by HGF appears to occur after NO-mediated signaling events.

Tatsumi and Allen (52) furthered our understanding of NO-mediated HGF release by

showing that the NO donor, sodium nitroprusside (SNP, 300 CIM), caused release of active HGF

from the extracellular matrix of rat EDL muscle and did not involve proteolytic cleavage of pro-

HGF. The addition of protease inhibitors to the incubation medium did not increase the amount

of pro-HGF released, demonstrating that the NO-stimulated mechanism of active HGF release

does not require proteolytic cleavage of pro-HGF. In addition, Yamada and colleagues (62)

provided evidence of the role of MMPs in the NO-dependent release of HGF from the

extracellular matrix.

More recently, Wozniak and Anderson (58) demonstrated that NO concentration regulates

the balance between satellite cell quiescence and activation on isolated fibers. A "normal"

concentration of NO maintains quiescence, while higher or lower levels of NO induce activation.









This was shown by stretching normal fibers and increasing NO concentration and satellite cell

activation. Likewise, satellite cell activation was increased in models of reduced NO availability,

namely fibers from mdx mice and NOS-I mice. These experiments highlight the importance of

NO for satellite cell activity.

Stretch

Mechanical stretch of cultured adult rat satellite cells was shown to cause release of HGF

and accelerated entry into the cell cycle (54). Conditioned media from cultures of stretched

satellite cells stimulated activation of unstretched cultures, and anti-HGF neutralizing antibodies

in conditioned media eliminated this response. Additionally, conditioned media from cells

stretched with L-NAME did not cause activation of unstretched satellite cell cultures, and HGF

was not released into the media. Media from unstretched cells cultured in the presence of an NO

donor was able to increase activation of control cultures, and HGF was detected in this media.

NOS activity assays indicated that NOS was stimulated when muscle satellite cells were

stretched. This data indicates that stretch stimulates intracellular events involving NO synthesis

and subsequent HGF release leading to satellite cell activation.

Mechanical stretch of single fibers has been used to activate satellite cells associated with

myofibers (59). Satellite cells on stretched fibers were significantly activated (incorporated

BrdU) at 30 minutes and 2 hours after initiation of stretch. It was speculated that these two peaks

may indicate that the same satellite cells entered proliferation after 30 minutes and 2 hours of

stretch and that each cell requires more than one stimulus to activate. Or, that the two peaks

symbolize two or more populations of satellite cells entering proliferation after differing stretch

stimuli (30 minutes and 2 hours). The latter explanation suggests that different regulatory events

may exist among various subpopulations of satellite cells.










This same study (59) also examined c-met expression in control, quiescent fibers. The

number of quiescent c-met' cells/fiber, determined using in situ hybridization with c-met mRNA,

was found to be approximately 1.4, with a range of 0-6 per fiber, in non-stretched cultures. There

are a number of c-met' cells that are not activated (BrdU-negative) by an activating stimulus

(HGF, CME, or stretch), indicating that the entire population of satellite cells is not uniformly

activated. This may be due to a subset of satellite cells that remain quiescent to maintain the

precursor pool. The use of stretch of single myofibers has the potential to further elucidate the

mechanisms of stretch-induced activation of satellite cells in their resident position and from a

quiescent state.

Mechanical stretch activates satellite cells on fibers (59) and in single cell cultures (54).

NOS activity is stimulated by stretch, and activation is NO-dependent (54). Likewise, HGF is

critical for activation and its stretch-induced release is prevented with NOS inhibition. Therefore,

NO appears to be an important signal for stretch-induced HGF release and satellite cell

activation.

Recovery from Atrophy

Skeletal muscle atrophies greatly in response to unloading. Two weeks of hindlimb

suspension resulted in a 36% reduction in myonuclear number in the mouse soleus (3 8). Two

weeks of recovery restored myonuclear number and muscle mass, suggesting that muscle

precursor cells can proliferate and fuse with myofibers. However, local gamma-irradiation,

which inhibits muscle precursor cell proliferation, prevented the recovery in muscle mass and

myonuclear number following unloading. Growth was normal in the first week of recovery, but

inhibited in the second week. These data indicate that proliferating muscle precursor cells are

required to completely restore myonuclear number and muscle mass after atrophy.









This same group showed that hindlimb suspension of two weeks reduced (by 14%) the

total number of cells isolated from atrophied muscles, and the number of satellite cells associated

with isolated myofibers compared to control (39). Atrophied myofibers produced fewer

myoD /desmin' cells per myofiber compared to non-atrophied fibers. To assess whether

proliferation was affected by hindlimb suspension, isolated precursor cells were labeled with

BrdU. A 25% decrease in BrdU /desmin' cells was found in atrophied muscles, suggesting the

ability to activate and/or proliferate was impaired. The ability of isolated cells from atrophied

muscles to differentiate and form myotubes was adversely affected by hindlimb suspension,

suggesting that differentiation and fusion were also affected. Conversely, recovery from atrophy

(normal ambulation) reversed all the effects of atrophy. Total cell number and myoD /desmin

cells per myofiber were increased. More BrdU /desmin+ cells were associated with recovered

muscles, and formation of large myotubes from muscle precursor cells was restored. Therefore,

no permanent changes occur in the muscle precursor pool with atrophic conditions. Upon

cessation of the atrophic stimulus, all changes were reversible.

Similarly, casting muscle for extended periods of time and then allowing for recovery

causes muscle wasting followed by muscle repair. Although a decline in satellite cell number is

evident with immobilization in the adult rat, remobilization leads to complete recovery of muscle

mass and myofiber regeneration (13). While protein synthesis increases during the recovery

phase, muscle precursor cell proliferation also increases to provide additional nuclei (35). The

satellite cells of adult animals are therefore able to activate and proliferate to promote recovery

of atrophic muscle.

Skeletal muscle has the unique ability to repair and regrow, or to hypertrophy with

appropriate stimuli. However, these actions cannot occur without the addition of new nuclei to









existing myofibers, or the fusion of myoblasts to form new myofibers. The source of these new

nuclei is the muscle precursor pool of satellite cells. Satellite cell activation is therefore an

indispensable step in muscle repair and adaptations to increased load. To date, HGF is the only

growth factor able to directly activate satellite cells, and its release is dependent on NO.

Aging and Muscle Satellite Cell Activity

Sarcopenia is the loss of skeletal muscle mass and strength with age. The age-related

muscle atrophy has been attributed to marked satellite cell quiescence (25). In aged muscle,

fewer satellite cells are present (5, 12, 28, 31, 45, 48, 51) and they exhibit a long latent period

between the activating stimulus and proliferation (53, 61). However, data suggest that aged

satellite cells retain their intrinsic myogenic potential, but the environmental stimuli are altered

(20, 5 1).

Satellite Cell Numbers with Age

Animal studies indicate that the satellite cell population declines with aging (5, 12, 28, 51).

However, results from human skeletal muscle are less harmonious. Many researchers report a

reduced proportion of human satellite cells with aging (31, 45, 48), while others report no

changes compared to young muscle (29, 47). Renault et al. (45) found the proportion of satellite

cells in the biceps brachii and masseter muscles was significantly lower in elderly men (mean

age, 74 years-old) compared to younger men (mean age, 23 years-old). Kadi et al. (31) studied

moderately active men and women, and found the elderly (age range, 70-83 years-old) had a

significantly lower number of satellite cells and proportion of satellite cells compared to young

subjects (age range, 20-32 years-old). Also, Sajko et al. (48) analyzed autopsy samples of vastus

lateralis muscle from young (mean age, 28.7 years-old) and old (mean age, 70.8 years-old) and

determined that numbers of satellite cells per fiber length and proportion of satellite cells were










significantly lower in the old muscles. These studies involving human subj ects support the

hypothesis that the satellite cell fraction declines with age.

In contrast, Hikida et al. (29) sampled young (mean age, 22.5 years-old) and elderly (mean

age, 65 years-old) men before and after a period of strength training, and found no significant

differences in the proportion of satellite cells between the age groups. Similarly, Roth et al. (47)

found no significant differences between the satellite cell population of young (20-30 years-old)

and old (65-75 years-old) men and women. Several factors may contribute to the opposing

results reported by various researchers. First, the number of individuals sampled and their ages

may affect the results. In general, age-related changes are clearly visible after age 70 (34). In the

study by Renault et al. (45), the mean age was 74 and 4 of the 6 subj ects were 78 years of age or

over. In contrast, the mean age of the elderly group was only 65 years in the study by Hikida et

al. (29). Other variables that may affect the differing results include different muscles sampled,

and the training status of individuals.

Most animal studies report declines in satellite cell numbers with age (5, 12, 28, 51).

Muscle-derived cell yield was significantly reduced in old rat muscle compared to young (5).

Bockhold and colleagues (12) reported a decline in the number of myogenic cells with aging in

the mouse using cells that migrated from single fibers in culture. Shefer and colleagues (51) used

molecular markers to characterize quiescent satellite cells and their progeny as they proliferate,

differentiate, and replenish the reserve pool in mouse muscle. The results of this study (5 1) show

that resident satellite cell numbers decline with age in myofibers from both fast- and slow-twitch

muscles. Enrichment of the culture medium with FGF2 enhances the regenerative capacity of

aged satellite cells, indicating the myogenic potential of aged satellite cells is not decreased as

abundance declines.









In contrast, Yablonka-Reuveni et al. (61) found that myofibers cultured from old rat

muscle retained a greater number of proliferating myoD' cells than young rat muscle. However,

the ages of old rats in this study were 9-11 months-old, and were compared to young (3 weeks-

old) rats in the rapidly growing phase. Conflicting data from various groups may result from

differences in species used, ages sampled, and techniques to identify satellite cells.

Aging and Muscle Regeneration

Following limb immobilization, young muscle has the ability to completely regain muscle

mass (13). In contrast, old muscle exhibits remarkably little regrowth following limb

immobilization (17, 43). The gastrocnemius of old rats showed no significant muscle regrowth,

even after 77 days of recovery. In addition, the soleus muscle of old rats fails to show any

regrowth after 30 days of recovery (43), whereas the soleus of young muscle recovers 37 and

40% of mass after 6 and 15 days of reload, respectively, after the same atrophy stimulus (18).

Chakravarthy et al. (17) administered insulin-like growth factor-1 (IGF-1) directly onto

atrophied muscle of 25-30 month-old rats, and found that in vivo skeletal muscle regrowth and in

vitro satellite cell proliferation were stimulated. They further showed that IGF-1 improves

proliferation by decreasing the cell cycle inhibitor p27Kipl1 via the PI3kinase/Akt pathway (16).

These data prompted the hypothesis that satellite cells were in sufficient quantity in old muscle,

but the systemic environment lacked appropriate growth factors/stimuli, or that old satellite cells

were less responsive to stimuli.

Age-related muscle loss could partially result from repeated episodes of incomplete repair

and regeneration (5). Muscle-derived cells (mdc) from young rats started to proliferate sooner

than adult and aged mdc. Cell cycle duration was faster in old cells compared to young, which

could lead to a more rapid exhaustion of replicative potential. Serum stimulation significantly

induced c-met and PCNA expression in young mdc, but the increase was much lower in old mdc.









When measured on crushed muscle extracts, plasminogen activators (PAs) and matrix

metalloproteinase 2-9 (MMP 2-9) enzyme activities were significantly lower with aging. These

modifications in events associated with satellite cell activation and proliferation may lead to

repeated bouts of incomplete repair and regeneration and to the loss of skeletal muscle mass with

agmng.

The hypothesis of incomplete repair by Barani et al. (5) is supported by others (26, 27).

Gallegly and colleagues (27) found evidence of ongoing regeneration in old rat muscle. Centrally

located nuclei were elevated in the soleus of aged (32 month-old) compared to young (6 month-

old) and adult (20 month-old) rats. Myonuclear domain size was decreased with age since soleus

muscle atrophy was not accompanied by a change in myonuclear number. Total BrdUT nuclei

were decreased with age, but BrdU+ myonuclei were increased. These results suggest that muscle

atrophy with age is coincident with increased myofiber nuclear density from proliferation and

fusion of satellite cells, resembling ongoing regeneration. Likewise, Edstrom and Ulfhake (26)

found no lack of regenerative drive in aged rat muscle. Aged soleus muscle had an accumulation

of nuclei, a wide distribution of fiber sizes, and an increase in embryonic MHC staining

compared to young muscle. The gastrocnemius muscle had high levels of MyoD and myogenin

mRNA expression compared to young. The regenerative drive is apparently intact in aged

muscle, but the efficiency or outcome of regeneration is poor.

In young rats, intermittent reloading for one hour/day during 7 or 14 days of hindlimb

suspension was sufficient to attenuate soleus muscle atrophy (27). However, this intervention did

not attenuate atrophy in aged (32 month-old) rats. Intermittent reloading was only associated

with an increase in BrdU' nuclei in young rats. This altered response of the soleus to an atrophy-

reducing stimulus likely results from altered satellite cell activation with age.









Conflicting data concerning myonuclear domain were published by Brack et al. (14). This

group found that loss of nuclei per unit of fiber length with age is exacerbated in larger fibers,

resulting in an increased domain size in these fibers, whereas Gallegly et al. (27) reported a

decrease in nuclear domain size. This loss of nuclei reported by Brack and colleagues (14) can be

accounted for by either excessive loss, or insufficient replacement. These researchers

hypothesize that muscle atrophy occurs to restore the nuclear domain size of a fiber to its set-

point after a transient increase in domain size due to nuclear loss/inadequate replacement. Thus,

the debate seems to be whether nuclei are lost prior to fiber atrophy, or vice versa.

Gene regulation during recovery from atrophy is altered in aged muscle (1, 27, 36, 43). In

particular, MyoD remained elevated in old muscle after regeneration, whereas its levels were

restored to normal in young animals (36). Plus, aging reduced the increase in MyoD protein

levels during hindlimb suspension (1). With advancing age, the increase in MyoD and myogenin

mRNA with hindlimb suspension in young rats was absent (27). Therefore, it is highly probable

that genes regulated by these transcription factors are dysregulated. Pattison et al. (43) also

demonstrated altered gene regulation with recovery from atrophy in aged muscle. A growth

factor (amphiregulin) was not upregulated, while a cell death-associated protein (clusterin)

remained upregulated in the old versus the young rat soleus muscle.

Notch Signaling and Satellite Cell Activation

Conboy and Rando (21) utilized an isolated myofiber model to study the role of Notch-1

and its antagonist Numb in satellite cell activation. Muscle injury tibialiss anterior) led to

increased expression of activated Notch-1 in myofibers isolated 72 hours after injury.

Interestingly, Numb immunostaining indicated an asymmetric distribution in myofiber-

associated mononucleated cells. Numb and pax3, a premyoblast marker, were virtually mutually

exclusive, whereas Numb mostly co-expressed with myf-5, pax7, and desmin. Therefore, Numb-









cells have a less committed muscle precursor phenotype, while Numb+ cells have a more

committed myogenic phenotype.

Expression of constitutively active Notch-1 in muscle progenitor cells increased pax3

expression and decreased myf-5, myoD, and desmin (21). Constitutively active Notch also

enhanced myoblast proliferation while reducing embryonic myosin heavy chain (MHC)

expression and the production of multinucleated myotubes. In contrast, overexpressing Numb

decreased pax3 expression and increased myf-5 and desmin protein levels. High levels of Numb

led to myotube formation. Taken together, these data indicate that Notch-1 activity promotes

myogenic cell proliferation, while inhibition of Notch-1 activity by Numb leads to exit from the

cell cycle, expression of muscle regulatory factors (MRFs), and myogenic differentiation.

The Notch-1 ligand, Delta, increases in response to muscle injury in satellite cells of young

(2-3 month-old) and adult (5-7 month-old) mice, but fails to do so in satellite cells of old (23-24

month-old) mice (19). Increased Delta expression in young and adult mice is coincident with

decreased Numb expression and increased proliferation. Satellite cells from old mice have

similar Notch-1 levels, but consistently lower levels of activated Notch than young mice.

Following muscle injury, Delta expression is upregulated in satellite cells from young mice, but

old satellite cells failed to do so. There was approximately one-fourth the number of activated

satellite cells in old muscle as in young and adult after injury. When Jagged-Fc, an inhibitor of

Notch activation, was introduced at injury sites, young muscle regeneration was ineffective,

similar to that seen in old muscle. Direct activation of Notch, using an antibody to its

extracellular domain, markedly improved in vivo regeneration in old muscle, making it similar to

young muscle. Therefore, Notch activation is not only necessary for young muscle regeneration

but is also sufficient to induce muscle regeneration in old muscle.










To further support these experiments, Conboy et al. (20) performed a series of parabiotic

pairings between young (2-3 month-old) and old (19-26 month-old) mice. These parabiotic

pairings allowed for a shared circulatory system, exposing old mice to factors present in young

serum and vice versa. After 5 weeks of parabiosis, the hindlimb muscles were injured and

inj ected with BrdU. Five days after injury, young muscle of both isochronic (young-young) and

heterochronic (young-old) pairings had regenerated. In contrast, old muscle from isochronic

pairings regenerated poorly. However, pairing with a young mouse significantly enhanced

muscle repair in old partners. This regeneration was due almost exclusively to activation of

resident aged satellite cells and not to infiltration of cells from the young partner. Satellite cells

from the aged partner in heterochronic pairings showed marked Delta upregulation, whereas this

induction was lacking in old isochronic pairings. There was a slight inhibition of Delta

upregulation in young satellite cells from heterochronic pairings compared to young isochronic

pairs.

The researchers also studied the effects of heterochronic parabiosis in vitro (20). Exposure

of satellite cells cultured from old muscle to serum collected from young mice improved Delta

upregulation and Notch activation compared to old satellite cells cultured in old serum. Young

serum also significantly increased myofiber-associated satellite cell proliferation of old cultures.

Young satellite cells exposed to old mouse serum exhibited reduced Delta upregulation. When

Notch signaling was inhibited, proliferation was decreased in both young and old satellite cells

cultured in young mouse serum. Taken together, these in vivo and in vitro data suggest that

factors in old serum negatively influence satellite cell activation, proliferation, and muscle repair.

However, components in young serum alone are sufficient to restore satellite cell function in

aged muscle. This implies that the old systemic environment is altered such that there is a









decrease in a positive factor found in young serum, an increase in a negative or inhibitory factor,

or both.

Nitric Oxide and HGF/c-Met in Aged Muscle

Neuronal nitric oxide synthase (nNOS) activity and percentage of NOS-containing fibers

were significantly reduced in muscles from aged rats compared to young (46). In young (8

month-old) animals, nNOS activity was highest in the EDL, which also had the greatest

percentage ofNOS-containing fibers. Activity was lowest in the soleus, and intermediate in the

diaphragm. NOS activity was significantly reduced in all muscles of the 24 month-old animals,

with EDL still having the greatest activity. In contrast to these results, Capanni and colleagues

(15) reported an increase in nNOS expression in 24 month-old rat gracilis muscle. However, this

group measured NO S expression via immunoblots and immunohi stochemi stry, whereas

Richmonds and colleagues (46) measured functional NOS activity. It is possible that NOS

enzymatic activity is inhibited even with an upregulation of nNOS protein expression.

Asymmetrical dimethylarginine (ADMA) is a likely candidate for the inhibition of NOS

activity with aging. ADMA is an endogenous inhibitor of NOS, and is elevated in the serum of

aged animals compared to young (60). The role of ADMA in endothelial dysfunction has been

studied (22), and by blocking NO generation, ADMA promotes processes leading to

atherogenesis, plaque progression, and plaque rupture. There are associations between elevated

levels of ADMA and many cardiovascular risk factors, which are also associated with advancing

age. Xiong et al. (60) measured ADMA levels in the serum of young (6 month-old) and old (20

month-old) rats. ADMA levels were significantly increased in the serum of the aged animals

compared to the young. Serum L-arginine levels were similar between the two ages, so the L-

arginine/ADMA ratio was lower in old rats compared to young. Lastly, serum concentrations of

nitrite were significantly decreased in old animals. Thus, it may be that ADMA levels increase









with age, and inhibit NO signaling in old muscle. To date, no studies have measured ADMA

levels in young and old skeletal muscle.

NO is important for triggering the release of HGF in the extracellular matrix to activate

satellite cells (2, 4, 50, 52). Barani and colleagues (5) reported a lower increase in the HGF

receptor, c-met, expression in cultured muscle-derived cells (mdc) of old rats with serum

stimulation compared to young. Young (3 week-old) and adult (9 month-old) mdc cultures

increased relative c-met expression by ~190% with serum stimulation. In contrast, old (24

month-old) mdc increased c-met expression by just 130% with serum stimulation. Cell surface

density of the c-met receptor was increased to the same extent in the three ages with serum

exposure.

In conclusion, muscle wasting associated with aging is well documented. Deficiencies in

satellite cell activation/proliferation, and incomplete repair of old muscle are also well

recognized. Several groups have identified strategies to improve muscle regeneration in aged

animals, including supplemental IGF-1 (17), forced Notch activation (19), and exposure to

young serum (20). A decrease in c-met expression and nNOS activity, and an increase in the

NOS inhibitor ADMA may contribute to the age-associated reduction in satellite cell activation.

Summary

Nitric oxide production triggering HGF release is an essential step in the activation of

satellite cells from a quiescent state. With aging, nNOS activity is decreased (46) and serum

levels of the NOS inhibitor ADMA are increased (60). It appears that aged satellite cells retain

the intrinsic ability to activate and proliferate when exposed to a young systemic environment

(21). This young environment both exposes aged satellite cells to young systemic factors, and

reduces their exposure to old systemic factors. Identifying those factors that affect the activity of

endogenous satellite cells will be crucial to improving the regenerative capacity of aged muscle.









It is possible that if satellite cell activation and proliferation can be maintained with age, skeletal

muscle loss due to sarcopenia will be prevented.









CHAPTER 3
MATERIALS AND METHODS

Experimental Designs

This proj ect was designed to answer the following questions with the accompanying

experimental designs:

Question 1. Is nitric oxide (NO) production reduced in old muscle?

Experiment 1. Extensor digitorum longus (EDL) muscles from young, adult, and old mice were

incubated in small tissue baths and NO production was measured using DAF fluorescence in the

presence and absence of electrical stimulation.

Question 2. Is there a deficit in satellite cell activation with aging in response to HGF?

Experiment 2. Young, adult, and old single myofibers were isolated from the gastrocnemius

muscle and cultured with different concentrations of HGF (1-50 ng/ml) to activate satellite cells.

Question 3. Is asymmetric dimethylarginine (ADMA) elevated in the muscle and/or blood of

aged animals?

Experiment 3. Blood was collected and serum isolated from young, adult, and old mice for

measurement of ADMA. Also, plantaris muscles from the three age groups were harvested and

homogenized for measurement of ADMA.

Question 4. Can ADMA inhibit satellite cell activation of young fibers?

Experiment 4. Gastrocnemius myofibers were isolated from young animals and exposed to

different concentrations of ADMA (0. 1-50 CLM).

Question Sa. Is there a deficit in satellite cell activation with aging in response to mechanical

stress?

Question 5b. Can L-arginine and/or a nitric oxide donor (DETA-NO) rescue satellite cell

activation in old muscle?










Experiments Sa and b. Isolated gastrocnemius myofibers from young, adult and old mice (4

mice per age group) were mechanically stimulated by centrifugation and activated satellite cells

were quantified by cell emanation as well as BrdU incorporation.

Animals

The subj ects were young (~2 mo old, n=13), adult (~10 mo old, n=8), and old (~22 mo old,

n=6) male C57 mice. All were housed in the J. Hillis Miller Animal Science Center in specific

pathogen-free housing. Animals consumed the same diet (mouse chow and water ad libitum),

and were maintained on a 12 h light: 12 h dark photoperiod. Mice were maintained in the facility

for at least one week prior to experiments. C57 mice are an accepted rodent model of aging and

were selected since our lab and others have successfully used them as subj ects for single fiber

and satellite cell isolation. All procedures were approved by the University of Florida' s Animal

Care and Use Committee.

Inz Vitro Electrical Stimulation and Measurement of Nitric Oxide Production

We validated a technique for quantifying the release of nitric oxide from isolated mouse

skeletal muscles at rest and immediately following contractions, using the cell impermeable

fluorescent detector of nitric oxide, 4,5-diaminofluorescein (DAF-2, Cayman Chemical

Company, Ann Harbor, MI). This method was first reported as a reliable method of quantifying

NO release from cultured endothelial cells (33). Addition of the NO-donor, MAHMA-NO (0-100

nM), to oxygenated buffer containing DAF-2 (0.1 CIM) was used to establish the relationship

between fluorescence intensity and NO concentration (R2=0.98, data not shown).

The EDL muscle from one leg was electrically stimulated for 10 min to quantify activity-

related nitric oxide production. The contra-lateral EDL was incubated without stimulation for the

same amount of time. Animals were anesthetized with inhaled isoflurane (2-5%) with oxygen as

the carrier gas. Both EDL muscles were removed and mounted in separate micro-tissue baths









between two platinum electrodes positioned 8 mm apart and surrounding the central part of the

muscle. Baseline tension was set for each muscle at 2.0 g of tension. During the experiment,

muscles were incubated in oxygenated Krebs-Henseleit bicarbonate buffer (KHB buffer: 1.2 mM

KH2PO4, 25 mM NaHCO3, 118.5 mM NaC1, 4.7 mM KC1, 0.25 mM CaCl2, 1.2 mM MgSO4, pH

7.4). The incubations were carried out at constant temperature (30oC) and under continuous

gassing with 95% 02/5% CO2.

After setting baseline tension (2.0 g), muscles were incubated for 10 min. Muscle viability

was indirectly assessed by testing whether muscles were able to contract in response to a specific

electrical stimulation protocol (1 train/sec, train duration of 250 ms, 50 Hz, 10 ms delay, 10 ms

duration, 30 V). Muscles were then electrically stimulated at 10 Hz, 50 V, 10 ms pulses with 90

ms delays for 10 min. while the contralateral EDL from each animal served as a non-contracting

control. Buffer was refreshed 3 times at the end of the contraction period, and muscles were

incubated with the cell impermeable fluorescent detector of nitric oxide, 4,5-diaminofluorescein

(DAF-2, 0.02 CLM, Cayman Chemical Company, Ann Harbor, MI) in the dark for 10 min.

Aliquots of buffer were analyzed for DAF fluorescence using a spectrofluorometer with

excitation wavelength at 485 nm and emission wavelength at 538 nm. Absorbances were

normalized to EDL wet weight.

To test for DAF-2 specificity, a separate experiment was conducted using both EDL and

soleus muscles from the same animal. In this experiment both EDL muscles were electrically

stimulated, while the soleus muscles were not. The protocol consisted of an initial 10 min

incubation, after which the inhibitor of NOS, L-NMMA (1 mM), was added to the buffer of one

EDL and one soleus. Muscles were incubated with L-NMMA for 10 min, followed by 10 min of

contraction (EDL muscles) or resting soleuss muscles). Buffer was refreshed 3 times afterwards,









followed by a 10 min incubation with DAF-2. Aliqouts of buffer were analyzed

spectrophotometrically for DAF fluorescence. We measured an ~10% increase in background

fluorescence in the soleus muscles induced by L-NMMA, and a total prevention of the

contraction-induced fluorescence in the EDL muscles with L-NMMA. Thus, DAF-2 specificity

was confirmed in this model.

Fiber Isolation

The gastrocnemius complex, consisting of the plantaris, soleus, and medial and lateral

heads of the gastrocnemius, was carefully removed from both hindlimbs. The plantaris and

soleus were removed, weighed, and frozen in liquid nitrogen for further analyses. The remaining

gastrocnemius muscle was rinsed in PBS, cut down the middle, weighed, and incubated in

DMEM containing 0.02% collagenase, type I (Worthington, Lakewood, NJ). Both gastrocnemius

muscles per animal were incubated in a 15-ml Falcon tube for 1.5 h at 37oC with gentle shaking.

After this incubation, the digest was carefully triturated three times, and incubated an additional

10 min. The collagenase was then diluted by allowing the digest to settle for approximately 5

min. Most of the media was removed and fresh media without collagenase was added. This was

repeated for a total of 3 times. The resulting digested muscles were decanted into p-100's

containing DMEM. Intact, isolated fibers were selected using a pipet. Only fibers that were free

of other cellular debris were selected and plated. These viable fibers were plated in 24-well

plates with growth factor-reduced matrigel (BD Biosciences, Bedford, MA) and serum free

media. Occasionally, emanating cells of non-muscle origin (non-spindle-shaped) were evident

and fibers containing these cells were excluded from analyses.

For the HGF experiment, fibers were cultured with 0.002% BrdU and HGF in serum free

media for 48 h, and fixed for satellite cell emanation quantification, and BrdU immunostaining.

For the ADMA experiments, fibers were incubated with ADMA at concentrations of 0. 1-50 CIM









for 24 h in serum free media. Then, 0.002% BrdU was added and the plates were centrifuged at

1500 x g for 30 min to induce satellite cell activation. The fibers were cultured for an additional

24 h and then fixed for quantification and BrdU immunostaining. For the final fiber experiment,

L-arginine and DETA-NO were added to the media with 0.002% BrdU immediately before

centrifuging at 1500 x g for 30 min to induce satellite cell activation. Fibers were cultured for 48

h and fixed for cell emanation quantification and BrdU immunostaining. A subset of fibers with

emanating cells was stained for myoD to confirm that emanating cells were of muscle origin.

Fibers stained for pax7 were plated in 24-well plates and fixed immediately.

Immunohistochemistry

To measure satellite cell activation, the number of cells staining positive for BrdU (BrdU )

were counted. Fixed fibers were incubated in 1 M HCI for 45 min at 45oC. This was neutralized

with 0.1 M borate buffer (pH 8.5) for 10 min. Fibers were blocked in 5% goat serum for 1 h,

followed by incubation in primary anti-BrdU antibody (1:75, Megabase Research, Lincoln, NE)

for 2 hours. They were then incubated in secondary anti-rabbit-Texas Red antibody (1:100,

Vector Laboratories) for 1 hour and stained with DAPI-containing mounting media (Vector

Laboratories). Fibers stained for pax7 were permeabilized with 0.1% Triton-X for 5 min. and

blocked in 5% goat serum for 1 h. Primary anti-pax7 (R&D Systems, Minneapolis, MN) was

applied for 1 h at 1:50 dilution. Secondary anti-mouse-Texas Red (1:100) was incubated for 1 h.

Fibers were mounted with DAPI-containing mounting media. All fibers were viewed on a Zeiss

microscope with rhodamine, FITC, and DAPI filters.

To confirm that the spindle-shaped emanating cells (i.e. mononuclear cells growing on

the substratum adj acent to myofibers 48 h after centrifugation) were of muscle origin, a subset of

cultures was stained for myoD using the Tyramide Signal Amplification (TSA) kit (Perkin

Elmer, Boston, MA). Fixed cultures were incubated in 3% H202 to quench endogenous









peroxidases. Fibers were blocked in TNB blocking buffer (0.1 M Tris-HC1, 0.15 M NaC1, 0.5%

Blocking Reagent, pH 7.5) for 1 h, followed by incubation in primary anti-myoD antibody

(1:200, Novo Castra, UK) for 2 h. They were then incubated in secondary anti-mouse-

biotinylated antibody (1:400, Pierce) for 1 h. Cultures were incubated with streptavidin-HRP for

30 min, followed by fluorophore tyramide working solution for 5 min. Finally, cultures were

mounted with DAPI-containing mounting media (Vector Laboratories). Greater than 95% of

these emanating cells were myoD confirming that the spindle-shaped cells adj acent to the

myofibers were emanating cells from those fibers.

Asymmetric Dimethylarginine (ADMA) ELISA

ADMA was measured in serum and muscle (plantaris) homogenate samples using an

ELISA kit (Alpco Diagnostics, Salem, NH) according to the kit instructions. The kit is a more

convenient method to measure ADMA than existing techniques, such as HPLC, liquid

chromatography-mass spectrometry, and gas chromatography-mass spectrometry, but correlates

well with these other methods (49).

Western Blotting

Plantaris muscle samples were homogenized in 50 mM Tris (pH 7.5), 150 mM NaC1, 5

mM EDTA, 50 mM sodium phosphate, 15% glycerol, ImM benzamidine, 10 mg/ml pepstatin A,

5 Clg/ml leupeptin, 1 mM PMSF, 2 CIM DTT, and 1 Clg/ml aprotinin. Homogenates were

centrifuged for 10 min at 1,000g to remove connective tissue and cellular debris and protein

content was quantified in the supernatant using the DC Protein Assay Kit (Bio-Rad Laboratories,

Richmond, CA). Forty micrograms of total protein were fractionated by SDS-PAGE and

electroblotted onto polyvinylidene difluoride (PVDF) membranes. Membranes were then

blocked for 1 h in Tris-buffered saline -Tween 20 (TBS-T) (Tris*HCI [pH 7.4], 150 mM NaC1,

0.1% Tween 20) containing 5% nonfat dry milk at room temperature, and then incubated with









primary antibody (nNOS 1:500, eNOS 1:500) overnight at 4oC. Membranes were washed three

times in TBS-T, 10 min each, followed by incubation with peroxidase-labeled anti-rabbit or anti-

mouse antibodies (Vector Laboratories, Burlingame, CA) for 1 h at room temperature. Reactions

were developed by using the enhanced chemiluminescence detection reagents (ECL Plus;

Amersham Biosciences, Buckinghamshire, UK), according to the manufacturer's instructions,

and protein levels were determined by densitometry (Scion Image software). Blots were stripped

(Restore Stripping Buffer, Pierce Chemical) and reprobed for p-actin (1:8000, Abcam) to control

for loading errors.

Statistical Analysis

Data were analyzed by a two-way ANOVA (age x treatment) with Tukey's HSD post hoc

test. Data from Experiment 4 (ADMA treatment of young fibers only), western blots, and pax7

data were analyzed by a one-way ANOVA. Significance was established a priori at p<0.05.

Values reported are means + SEM.









CHAPTER 4
RESULTS

Muscle Atrophy in Male C57 Mice

We found evidence of both maturation and senescence effects when comparing muscle

masses and the ratio of muscle mass to body mass between our three age groups (Table 4-1).

Specifically, muscle masses for the gastrocnemius, plantaris, soleus, and tibialis anterior (TA)

significantly increased between ages 2 and 10 months, representing maturation effects. The

muscle mass to body mass ratio for the gastrocnemius, plantaris, and TA significantly decreased

between these ages. Senescence effects were noted as a significant decrease in gastrocnemius,

plantaris, and soleus masses between ages 10 and 22 months. In addition, the muscle mass to

body mass ratio of the soleus significantly decreased between ages 10 and 22 months. The total

protein per plantaris muscle was significantly increased from 2 month to 10 months, while total

protein per gram of wet weight was not different between ages.

Nitric Oxide Production from Contracting Muscle

Since nitric oxide (NO) is a primary signal for satellite cell activation (2, 4, 50, 52), we

sought to determine whether NO production was reduced from aged muscle and whether this

could account for the previously reported deficits in satellite cell activity (53, 61) and muscle

repair (17, 19, 20, 43). NO production, as quantified by DAF fluorescence immediately

following a 10-min contraction protocol, was significantly lower from adult and old EDL

muscles as compared to young muscles (Figure 4-1). NO production did not differ between adult

and old EDL muscles. This finding was unexpected since deficits in satellite cell activity are not

evident in the adult muscles, compared to young. All ages exhibited a significant increase in NO

production following contraction, and the fold change over resting was not significantly reduced









in aged and adult EDL muscles compared to young, suggesting that NO production is not

limiting in adult or aged muscles.

Nitric Oxide Synthase Protein Expression Is Not Reduced

Next, we sought to determine whether protein expression of nitric oxide synthases (NOS)

was decreased in aged muscle. Both constitutively active NOS isoforms, nNOS and eNOS, were

expressed equally in plantaris homogenate from young, adult, and aged muscle (p = 0.907 for

nNOS and p=0.976 for eNOS, Figures 4-2 and 4-3, respectively). Thus, a reduction in NOS

protein content cannot explain the decreased NO production from adult and aged muscle.

Asymmetric Dimethylarginine Can Inhibit Satellite Cell Activity

We found that inhibition of NOS activity with the competitive inhibitor, asymmetric

dimethylarginine (ADMA), prevented satellite cell activation, as quantified by both satellite cell

emanation (Figure 4-4A) and BrdU incorporation (Figure 4-4B), from young mouse myofibers

(Figure 4-5A-D). In all isolated myofibers experiments, satellite cell activation was assessed by

quantifying emanating cells, and positive immunostaining for BrdU. In a subset of fibers with

emanating cells, greater than 95% of these cells were myoD confirming that the spindle-shaped

cells adj acent to the myofibers were emanating from those fibers. ADMA doses of 0. 1-50 CLM

reduced satellite cell activation of young myofibers exposed to the inhibitor for 24 h prior to

mechanical stimulation. Both 10 and 50 CLM ADMA significantly reduced satellite cell

emanation from young fibers (Figure 4-5A), while 50 CLM ADMA significantly reduced BrdU

incorporation compared to control (Figure 4-5C). However, even the lowest dose (0.1 CLM)

negatively affected satellite cell activation on young fibers. Our findings suggest that inhibition

of NOS activity with the competitive inhibitor, ADMA, is sufficient to attenuate satellite cell

activation from young fibers exposed to mechanical stress.









Asymmetric Dimethylarginine Is Not Elevated in Aged Muscle and Blood

Next, we measured the levels of endogenous asymmetric dimethylarginine (ADMA) in

the serum and plantaris homogenate of young and old animals. ADMA levels in the muscle and

serum did not differ between young and old mice (Table 4-2), thereby eliminating an

accumulation of ADMA as a potential cause of decreased NO production with age.

Aged Myofibers Have Equal Numbers of Pax7+ Satellite Cells

We quantified the number of pax7+ nuclei/Hiber by immunostaining myofibers

immediately Eixed after isolation and plating (Figure 4-6). There were no significant differences

between the mean number of pax7+ nuclei per fiber for any age group (Young = 3.16 & 0.296,

Adult = 3.08 & 0.3 84, Old = 2.79 & 0.321). This is important because it confirms that any deficits

in satellite cell activation are not due to fewer satellite cells present initially on aged fibers.

Satellite Cell Response to HGF Is Not Altered with Age

To determine whether the deficit in aged satellite cell activation is upstream of HGF

release, we examined the response of satellite cells on isolated myofibers to various doses of

HGF. Adult and aged satellite cells reacted similarly to young satellite cells in response to HGF

(Figure 4-7A-D). Regardless of age, 10-20 ng/ml of HGF significantly increased satellite cell

activation (as quantified by satellite cell emanation and BrdU incorporation 48 h post-plating).

Fifty ng/ml HGF did not significantly increase BrdU incorporation above control levels for any

age. This finding implies that there is an upper limit to HGF's effects on satellite cell activation.

However, satellite cells from adult and aged fibers respond to HGF in the same manner as young

satellite cells, suggesting that the aging deficit lies upstream of HGF release.

L-Arginine and a Nitric Oxide Donor Can Rescue Aged Satellite Cell Activity

The similar response of satellite cells to an HGF dose response suggests the aging deficit

lies upstream of HGF release. Since NO triggers HGF release, we sought to determine whether









the NOS substrate, L-arginine, or a NO donor (DETA-NO) could "rescue" satellite cell

activation from mechanically stimulated aged myofibers. Myofibers were treated with L-arginine

(2 mM) or DETA-NO (10 pLM) and centrifuged to stimulate activation of satellite cells. Adult

satellite cell activation (satellite cell emanation and BrdU incorporation 48 h post-plating)

mirrored young satellite cell activity with both treatments. Satellite cell activation from aged

myofibers was improved with L-arginine and DETA-NO (Table 4-3 and Figure 4-8 A-D).

However, both treatments increased satellite cell activity in young and adult fibers as well.

Importantly, young, adult, and old control fibers, in the absence of mechanical stress, were not

significantly different when comparing satellite cell emanation or BrdU incorporation. There was

a trend for centrifugation to stimulate satellite cell emanation in young (p=0.074) and adult

(p=0.065) fibers, while this was not true for old fibers (p=0.788).

To confirm these results were due to NO production and not indirect effects of the parent

compound, we tested a dose response of NO-depleted DETA (5-50 pLM) and found no increase in

satellite cell activation above control levels (data not shown). In sum, both the NOS substrate

and the NO donor were able to rescue satellite cell activity from aged muscle to above young

control levels, but were not able to normalize aged satellite cell activity to levels measured in

young myofibers with the same treatment.


















126.92 & 2.45
5.13 & 0.069

17.88 & 0.49
0.72 & 0.016
4.58 & 0.143
0.86 & 0.046

10.02 & 0.32
0.40 + 0.011

49.98 & 1.64
2.02 & 0.060


Table 4-1. Body mass, gastrocnemius mass, muscle mass/body mass ratios, and total protein for
young, adult, and old mice. Values represent mean & SEM. Total protein
measurements were made for plantaris muscle.* Significantly different from Young.


'Significantly different from Adult.
Young (2 mo)


24.77 & 0.58


Adult (10 mo)
34.63 A 1.38*

160.40 & 2.14*
4.67 & 0. 117*

21.58 & 0.50*
0.63 & 0.026*
5.06 & 0.236
1.07 & 0.055*

13.55 & 0.45*
0.40 + 0.018

59.28 & 0.86*
1.73 & 0.057*


Old (22 mo)
29. 17 & 0.95*

131.05 & 2.887#
4.50 + 0.063*

18.21 & 0.46#
0.63 & 0.013*
5.00 + 0.349
0.93 & 0.088

9.61 & 0.34#
0.33 & 0.014*

55.68 & 1.61*
1.92 & 0.067


Body Mass (g)
Gastrocnemius
Mass (mg)
Mass/Body Mass
Plantaris
Mass (mg)
Mass/Body Mass
Protein (mg/g w.w.)
Total Protein (mg)
Soleus
Mass (mg)
Mass/Body Mass
Tibialis anterior
Mass (mg)
Mass/Body Mass










Table 4-2. Asymmetric dimethylarginine (ADMA) concentration (Cpmol/L) in serum and muscle
of young and old mice. Values represent mean & SEM. Muscle ADMA was measured
in plantaris homogenate.
Young (2 mo) Old (22 mo)

Serum 1.46 & 0. 14 0.82 & 0.15

Muscle 6.18 & 0.87 5.00 + 0.44





Table 4-3. Quantification of satellite cell activation as measured by (A) cell emanation and (B)
BrdU incorporation from young, adult, and old myofibers 48 h post-plating. Values
represent mean & SEM for n = ~50-90 fibers/group. Centr.=Centrifuged for 30 min at
1500g. *Significantly different from Young Control and Young Centrifuged.
#Significantly different from Young Centrifuged + Arginine. tSignificantly different
from Young Centrifuged + DETA-NO. ~Significantly different from Age-Matched
Centrifuged. mSignificantly different from Adult Control. &Significantly different from
Adult Centrifuged. 'Significantly different from Adult Centrifuged + Arginine.
sSignificantly different from Adult Centrifuged + DETA-NO. KSignificantly different
from Old Control.
A. Young (2 mo) Adult (10 mo) Old (22 mo)


Control


0. 18 & 0.059


0. 16 & 0.056

0.42 & 0.069#,?

1.18 &0.115" CK

1.38 & 0.111''f"'*,,K


0.19 & 0.088

0.24 & 0.072#t'5

0.74 & 0.103*,#,tt, m,,v,5,K

0.73 & 0.091*#,tf -m,,v,5,K


Centrifuged

L-Arg + Centr.

DETA-NO + Centr.


0.42 & 0.068

1.04 & 0.101 ,OK

1.30 + 0.101 ,OK


Young (2 mo)

0.21 & 0.08

0.28 & 0.061

0.60 + 0.079

0.79 & 0.085*,0,K


Adult (10 mo)

0.26 & 0. 11

0.27 & 0.065?

0.56 & 0.084

0.73 & 0.089*,t,0,K


Old (22 mo)

0.22 & 0.09


Control


0. 19 & 0.058#,tM,!i

0.44 & 0.087t

0.60 + 0.083~


Centrifuged

L-Arg + Centr.

DETA-NO + Centr.




















*,#,+ *~#,+


Resting Contracting


Figure 4-1. Nitric oxide (NO) production as quantified by 4,5-diaminofluorescein (DAF)
fluorescence following 10 min of in vitro EDL muscle contraction. Values are
normalized to EDL muscle wet weight. Values represent mean + SEM. *Significantly
different from Young Resting, p<0.05. #Significantly different from Age-Matched
Resting, p<0.05. 'Significantly different from Young Contracting, p<0.05.


BBB~~ oung
BEWAdult
I IOld














A. nNOS


Young


Adult


Old







Old


Young Adult


Beta-actin


1.
*







O


Figure 4-2. Protein expression of neuronal nitric oxide synthase (nNOS). (A) Representative
immunoblot of nNOS from young, adult, and old plantaris muscle. Beta-actin served
as a loading control. (B) Quantification ofimmunoblots for nNOS normalized to
beta-actin. Values represent mean a SEM.


.I~c--"~ I~e" :





-- .


A. eNOS


Young


Adult


Beta-actin


Young Adult


Old


1.0
a .5



0.0.
*
O


-r


Young


Figure 4-3. Protein expression of endothelial nitric oxide synthase (eNOS). (A) Representative
immunoblot of eNOS from young, adult, and old plantaris muscle. Beta-actin served
as a loading control. (B) Quantification of immunoblots for eNOS normalized to beta-
actin. Values represent mean + SEM.








































B.l~













Figure 4-4. Representative images of activated satellite cells. (A) Representative light image of a
satellite cell emanating from an isolated mouse myofiber indicated by white arrow
(120x magnification). (B). Representative fluorescent image of a satellite cell staining
positive for BrdU (red). Nuclei are stained blue with DAPI (60x magnification).























9. B.





S0.45 ( -1----- 7

z II C .30---
+ 0.25~k 2~TTr"~B IIII ~


OgM 0.1B88p M 1.0p 10 50M2 OH 01M .g 0M 5p
ADM Cocetato a. ADM Concentrati
C., D..





pecnage ~ofHenrs ithBrd nule folw ngxosure toa rigcncentrations
ofAD A24hpro t ecancl etrfgain.Vlus ereet en EMo


~50 fiesgop Sgienl ifrn rm0p D A
































Figure 4-6. Representative image of a fiber stained for pax7 (red) immediately following
isolation and plating (~30 fibers per age group). White arrows indicate pax7' nuclei.
Blue DAPI-stained nuclei, Red pax7' nuclei (60x magnifieation).
















+Y'oung
*Adul


HGF Dose (nglml)


+Y~oung
+Add t
* Oki


20
HGF Dose (nglml)


Figure 4-7. Response of fibers to hepatocyte growth factor (HGF). (A) Mean satellite cell
emanation per fiber and (B) percentage of fibers with emanating satellite cells in
response to varying concentrations of HGF. (C) Mean BrdU+ cells per fiber and (D)
percentage of fibers with BrdU+ nuclei following exposure to varying concentrations
of HGF. Approximately 50 fibers/group were analyzed.


SYoung
BAdul


HGF Dose (ngiml)


B Young
MAdull
tlkI


HGF Dose (ngld)











*,+,
#.5
t,+,


MControl
155centrifugation
ESCentrifugation + L-arginine
[ESCentrifugation + DETA-NO


M Control
M Centrifugation
ECentrifugation +
mmCentrifugation +


L-arginine
DETA-NO


Figure 4-8. Response of fibers to L-arginine and DETA-NO. (A) Mean satellite cell emanation
per fiber and (B) percentage of fibers with emanating satellite cells in response to
treatment with 2 mM L-arginine or 10 CLM DETA-NO and centrifugation (1500g, 30
min). (C) Mean BrdU' cells per fiber and (D) percentage of fibers with BrdU' nuclei
following exposure to L-arginine and DETA-NO and centrifugation. Approximately
90 fibers per group were analyzed. (A.) and (C.) represent mean + SEM.
*Significantly different from Young Control. +Significantly different from Age-
Matched Control. #Significantly different from Young Centrifuged. SSignificantly
different from Age-Matched Centrifuged.
































Young


AdulIt


11(1


#k g 1--1 I ll 5


gill 1~~111


Y


_


Figure 4-8 Continued.


M Control
M Centrifugation
I SCentrifugation + L-arginine
[EE]Centrifugation + DETA-NO










M Control
MB Centrifugation
I|||||Centrif ugation + L-arginine
[mICentrifugation + DETA-NO


Young


AdulIt


1


111









CHAPTER 5
DISCUSSION

Main Findings

Although our old mice were in the early stages of senescence, based on survival curves

for this strain, we found evidence of muscle atrophy in the gastrocnemius, plantaris, and soleus

muscles. Single myofibers were isolated from the gastrocnemius muscles with resident satellite

cells remaining. We studied the activation of these satellite cells, defined as myogenic cell

emanation from the fiber and BrdU incorporation at 48 h post-plating. The main findings of this

study are: 1.) Satellite cell activation in response to mechanical stimulation is reduced with age;

2.) Nitric oxide (NO) and L-arginine supplementation effectively increase satellite cell

activation; however, an age-related deficit remains; 3.) In vitro NO production is reduced from

adult and aged contracting muscle; and 4.) Aged fibers respond normally to exogenous

hepatocyte growth factor (HGF). The data indicate that the aging deficit in satellite cell

activation lies in the sensitivity of aged satellite cells to NO.

Reduced Satellite Cell Activation in Response to Mechanical Perturbation

In response to a mechanical stress (centrifugation), the increase in dividing cells emanating

from aged fibers was reduced compared to young, and fewer nuclei stained positive for BrdU

(Table 4-3 and Figure 4-8 control vs. centrifuged, across supplement groups). These data depict

the deficit in satellite cell activation from old muscle. Nevertheless, there were no differences in

pax7' satellite cells present on young, adult, and old fibers fixed immediately after isolating, in

agreement with several human (29, 47) and animal studies (42). In contrast, many animal studies

indicate that the satellite cell population declines with aging (5, 12, 28, 51). Differences in

muscles studied, ages of animals, and methods for identifying satellite cells may explain the

differences in findings. Unlike most aging studies, our old group (22 mo) was younger than the









50% mortality age for this strain (27 mo). Therefore, it is possible that the decline in the

quiescent satellite cell population reported by other authors occurs only during advanced

senescence. Our animals, which were in early senescence, exhibit impaired satellite cell

responsiveness to external stimuli, without a decline in the overall quiescent population.

Our data imply that there is a deficit in the signaling pathways leading to activation of

resident satellite cells on old muscle. This pathway involves a sequence of events including: 1)

mechanical perturbation, 2) nitric oxide release, 3) HGF release from the extracellular matrix, 4)

HGF binding to the c-met receptor on satellite cells, and finally, 5) activation of DNA synthesis

and migration of satellite cells (Figure 5-1). We studied the mechanism of age-associated

satellite cell dysfunction by examining satellite cell activation in isolated myofibers following

experimentally induced mechanical perturbation (centrifugation), exogenous NO treatment, or

exogenous HGF application, in order to localize the aging deficit.

L-Arginine and a Nitric Oxide Donor Can Rescue Aged Satellite Cell Activity

The NOS substrate, L-arginine, or an NO donor, DETA-NO, "rescued" satellite cell

activation in mechanically stimulated aged myofibers, in that satellite cell activation from old

fibers exposed to these treatments exceeded the level of satellite cell activation in young control

fibers (Table 4-3 and Figure 4-8). We confirmed that the DETA-NO effects were due to NO

production and not indirect effects of the parent compound, DETA, by exposing fibers to NO-

depleted DETA (5-50 pLM). These data suggest that an age-related reduction in NO production

could contribute to reduced satellite cell activity. However, both L-arginine and DETA-NO

treatments also increased satellite cell activity in young and adult fibers such that satellite cell

activity in old fibers remained significantly lower than in young and adult fibers with the L-

arginine or DETA-NO treatments. It appears that NOS activity/NO production may limit satellite









cell activation from all ages since exogenous NO supplementation increased satellite cell activity

across age groups.

Our centrifugation protocol was a submaximal stimulus that did not induce 100% satellite

cell activation. Consequently, the degree of satellite cell activation following a fixed stimulus

(i.e., centrifugation or exposure to a Eixed dose of DETA-NO or L-arginine) indicates the

sensitivity of the system to that particular stimulus. Therefore, in spite of the potential

therapeutic effects of augmenting NO production, we must conclude that age-related deficits

responsible for compromised satellite cell activation lie downstream of this event.

Other groups have completely restored satellite cell activation/proliferation in old muscle

with exposure to young serum (20), forced Notch activation (19), IGF-1 (17), or basic FGF2

(51). However, these researchers studied both activation and proliferation. Our experimental

design, on the other hand, focused primarily on the activation step, since fibers were Eixed before

significant proliferation occurred. We conclude that the aging deficit in satellite cell activation

lies in the sensitivity of aged satellite cells to NO.

Nitric Oxide Production and NOS Protein Expression from Aged Muscle

Nitric oxide is a primary signal for HGF release leading to satellite cell activation (2, 4, 50,

52). Therefore, we hypothesized that NOS expression and/or NO production would be reduced in

aging skeletal muscle. Myofiber isolation required both gastrocnemius muscles, leaving

insufficient tissue for additional measurements. Therefore, in vitro contraction-induced NO

production was measured in the EDL, and NOS protein expression in the plantaris. These

muscles were chosen since they resemble the gastrocnemius in fiber type (primarily type II) and

location (hindlimb). NO production, as quantified by DAF fluorescence following muscle

contraction, was significantly lower in aged and adult, compared to young EDL muscle (Figure

4-1). While all ages exhibited an increase in NO production following contraction, the young









EDL muscles produced significantly more NO following contraction than adult or aged muscles.

However, the fold-change over resting values was not significantly reduced in adult or aged

muscles, suggesting that contraction-induced NO production is not limiting in aged animals.

Ours is the first study providing evidence of a functional decrease in NO production from

aged, as well as adult muscle. The reduced NO production from adult muscle was unexpected

since no reduction in satellite cell responses were noted in adult fibers compared to young

(Figure 4-8). We found no differences in nNOS and eNOS protein expression between young,

adult, and old plantaris muscles (Figures 4-2, 4-3). Thus, down-regulation of NOS enzyme

expression may not explain the reduced NO production from adult and old EDL muscles. We did

not measure NOS activity, but Richmonds and colleagues (46) found a reduction in nNOS

activity and percentage ofNOS-containing fibers in muscles from aged rats (24 mon) compared

to young (8 mon). In contrast, Capanni and colleagues (15) reported an increase in nNOS

expression in 24 month-old rat gracilis muscle. However, this group measured NOS protein

expression via immunoblots and immunohistochemi stry, whereas Richmonds and colleagues

(46) measured functional NOS activity. It is possible that NOS enzymatic activity is inhibited

even with an upregulation of nNOS protein expression (15), or no change in NOS expression as

we show.

While it is an interesting observation that NO production is decreased in adult and aged

muscle compared to young, it does not appear that a significant loss of contraction-induced NO

production by the EDL is associated with senescence (i.e., no difference between adult and old

muscles). Therefore, reduced NO production is unlikely to explain age-related deficits in satellite

cell activation in these muscles.









Satellite Cell Response to HGF Is Not Altered with Age

We determined that the deficit in aged satellite cell activation lies upstream of HGF release

since the response of aged satellite cells on isolated myofibers to a dose response of HGF was

similar to young satellite cells. Regardless of age, 10-20 ng/ml of HGF significantly increased

satellite cell activation (satellite cell emanation and BrdU incorporation 48 h post-plating), in

agreement with previously published studies (55, 58). Fifty ng/ml HGF did not significantly

increase BrdU incorporation above control levels for any age. This finding implies that there is

an upper limit to HGF's effects on satellite cell activation.

The similar response of young, adult, and old satellite cells is important because it implies

that all events downstream of HGF release, such as HGF/c-met colocalization and c-met

signaling, are normal in aged muscle and satellite cells. In contrast, Barani and colleagues (5)

reported a lower increase in the HGF receptor, c-met, expression in cultured muscle-derived cells

(mdc) of old rats with serum stimulation compared to young. Young (3 week-old) and adult (9

month-old) mdc cultures increased relative c-met expression by ~190% with serum stimulation.

In contrast, old (24 month-old) mdc increased c-met expression by just 130% with serum

stimulation. However, the cell surface density of the c-met receptor was increased to the same

extent in the three ages with serum exposure. Therefore, similar responses to a fixed dose of

HGF, as we observed, are not surprising. These observations effectively localize the aging deficit

in satellite cell activation to the mechanisms mediating HGF release in response to NO

production.

Asymmetric Dimethylarginine Is Not Elevated in Aged Muscle and Blood but Does Inhibit
Satellite Cell Activity

To explain the aging deficit in satellite cell activation and the reduction in NO production

with age, we studied the levels of ADMA, a competitive inhibitor of NOS, in the serum and









plantaris homogenate of old and young mice, and its effects on satellite cell activity. Conboy and

colleagues (20) used a series of in vivo and in vitro experiments to show that factors in old serum

negatively influence satellite cell activation, proliferation, and muscle repair. However,

components in young serum alone are sufficient to restore satellite cell function in aged muscle.

Their data imply that the old systemic environment is altered such that there is a decrease in a

positive factor found in young serum, an increase in a negative or inhibitory factor, or both. We

hypothesized that ADMA was that negative factor found at high levels in old serum. As a NOS

inhibitor, it may potentially inhibit satellite cell activity.

We found that ADMA doses of 0. 1-50 CLM negatively impacted satellite cell activation

(Figure 4-5). Myofibers from young muscles were adversely affected by ADMA such that 10

and 50 CLM significantly reduced satellite cell emanation from fibers, while 50 pLM ADMA

significantly reduced BrdU incorporation compared to control. Our findings provide further

evidence of the importance of NO for satellite cell activation since ADMA is sufficient to

attenuate satellite cell activity.

However, we did not measure an increase in ADMA in old animals. ADMA levels were

similar in the serum and muscle homogenate of young and old animals (Table 4-2), suggesting

that ADMA concentration and subsequent NOS inhibition is not elevated with age.

One group of researchers has reported an increase in ADMA in serum from aged (20 mo)

rats compared to young (6 mo) animals (60). Serum L-arginine levels were similar between the

two ages, so the L-arginine/ADMA ratio was lower in old rats compared to young. Lastly, serum

concentrations of nitrite were significantly decreased in these old animals. While ADMA is an

appealing candidate for the reduction in NO production with age, we were unable to provide

evidence of an accumulation of ADMA in old mice. Nevertheless, our data showing that ADMA









inhibits satellite cell activity on fibers confirm the integral role of NO signaling for satellite cell

activation from quiescence.

Response to Nitric Oxide

Under conditions where satellite cells are kept quiescent, muscle produces NO in low

level pulses (56, 58). On the other hand, mechanical stress, associated with satellite cell

activation, produces a bolus of NO via nNOS acting as a mechanotransducer (56). NO is

important for triggering the release of HGF from the extracellular matrix (ECM) to activate

satellite cells (2, 4, 50, 52). Tatsumi et al. (54, 55) have demonstrated that release of HGF, in

response to mechanical perturbation, is NO-dependent. When released, HGF rapidly co-localizes

with its receptor on satellite cells, the c-met tyrosine kinase receptor, and triggers activation from

quiescence (53). Once activated, satellite cells synthesize both HGF and c-met (3, 53). HGF is

synthesized as a precursor protein requiring proteolytic cleavage to produce the active growth

factor. However, Tatsumi and Allen (52) confirmed that active heterodimeric HGF is present in

the ECM of uninjured rat muscle, indicating that proteolytic cleavage to activate HGF is not a

rate-limiting step in satellite cell activation, but rather the steps leading to release of active HGF

from its ECM tethering.

Yamada and colleagues (62) recently provided evidence of the involvement of matrix

metalloproteinases (MMPs) in the NO-dependent release of HGF in vitro. Inhibition of MMPs

along with treatments that induce satellite cell activation (stretch, NO supplementation) inhibited

activation of cultured rat satellite cells. In addition, conditioned medium from satellite cells

exposed to activating stimuli plus the inhibitor of MMPs did not activate cultured satellite cells

and did not contain HGF. Importantly, the MMP inhibitor did not inhibit NOS activity. These

data suggest that MMPs cleave HGF from the ECM and that this step is triggered by NO release.









Barani et al. (5) measured MMP 2-9 activity on crushed muscle extract (CME), and

found that these activities were reduced with aging. MMP2-9 enzyme activity measured in initial

muscle derived cells (mdc) was identical in young (3 week-old) and old (24 month-old) rats, and

serum exposure increased activity uniformly in both ages. However, when measured on CME,

MMP 2-9 enzyme activity was ~2-fold lower in old CME compared to young. This may explain

the aging deficit in satellite cell activation and warrants further investigation. Another possible

explanation for the aging deficit in satellite cell activation is a decrease in endogenous HGF

levels with aging. If HGF production goes down with age, there would be less present to

stimulate activation in response to NO signaling. Thus, we postulate that an age-related decline

in MMP activity and/or a reduction in HGF expression decreases the sensitivity of satellite cells

to NO, thus limiting satellite cell activation.

Limitations and Future Directions

The age of the "old" mice (22 mo) may have limited the severity of the aging deficit in

satellite cell activation in response to mechanical perturbation. According to the National

Institute on Aging (NIA), the age of 50% mortality for male C57 mice is 27 months. Using mice

27 months or older may have amplified the effects of our treatments on satellite cell activity. On

the other hand, the presence of a deficit in satellite cell activation during early senescence (i.e.,

22 mo) highlights the potential clinical significance of this phenomenon in an aging population.

Higher doses of arginine and DETA-NO may have improved satellite cell activation from

old fibers. These doses were selected based on dose responses in young muscle. It may be that

higher doses were necessary to maximally stimulate satellite cell activation on old fibers. Plus,

our mechanical stimulus (centrifugation, 1500g, 30 min) was a submaximal stimulus for

activation. Using a more severe mechanical perturbation and/or higher doses of supplements may

have activated more satellite cells and eliminated aging deficits. Nevertheless, the submaximal









stimuli chosen allows assessment of the sensitivity of the satellite cell activation system. Future

studies should focus on physiologically-relevant activating stimuli, such as stretch or injury.

Isolated myofibers are extremely useful for studying satellite cells in their resident

position on the myofiber. However, it is difficult to harvest fibers for such assays as Western

blotting or PCR. Thus, we were limited to immunostaining of fibers to identify and count

satellite cells. Future studies should include treatment of the whole animal with L-arginine or NO

donors to allow for more robust measurements.

We did not measure NOS activity in the gastrocnemius to confirm that it is reduced with

aging. NOS protein expression in the plantaris was not changed with aging, but NO production

was decreased in contracting adult and aged EDL muscle. We cannot determine whether NO

production by isolated gastrocnemius myofibers during centrifugation was reduced. Regardless,

reduced NO production does not seem to be the cause of the aging deficit in satellite cell

activation since this deficit persists with administration of a fixed amount of NO donor.

Lastly, we did not find a reduction in pax7' cells per fiber between our ages. However,

due to the limitation of available fibers, only ~30 fibers from each age were stained and

analyzed. Although there did not appear to be a trend for reduced satellite cells in the aging

gastrocnemius, the variability between fibers within an age group, and even within one mouse,

was high. Staining more fibers, such as 100 fibers per age group, may have shown a significant

loss of satellite cells with age. Future studies need to confirm equal pax7+ nuclei per fiber with

this strain of mouse and these ages.

Conclusions

We have successfully narrowed the aging deficit in satellite cell activation in response to

mechanical perturbation to the steps between NO production and HGF release. Based on our

data, NO production does not limit satellite cell activity in old muscle, and the steps downstream









of HGF are not impaired in old fibers. Figure 5-1 diagrams the signaling events leading to

satellite cell activation from quiescence. We conclude that the deficit in activation lies between

NO production and HGF release, in the sensitivity of old satellite cells to NO.








Mechanical Perturbation


SNO Release

l(Location of Aging Deficit)
HGF Release from ECM


HGF/c-met co-localization and Signaling

Satellite Cell Activation
(DNA Synthesis and Migration)

Figure 5-1. Overview of the steps leading to satellite cell activation and the potential location of
the aging deficit.









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BIOGRAPHICAL SKETCH

Jenna Betters was born in New Smyrna Beach, Florida, in 1979. She was salutatorian of

her high school graduating class in 1997. She received her bachelor' s degree in biology in 2001

from the University of North Florida in Jacksonville, Florida, where she was a member of Phi

Kappa Phi Honor Society, University Scholar's Honor Society, and Golden Key International

Honor Society. Jenna obtained her Master of Science in Exercise and Sport Sciences from the

University of Florida in 2004. Her thesis was titled "Trolox supplementation during mechanical

ventilation attenuates contractile dysfunction and protein degradation." She received a Graduate

Alumni Fellowship, allowing her to pursue the Doctor of Philosophy degree. She has worked as

a research assistant in the Molecular Physiology Laboratory within UF's Center for Exercise

Science since 2001. After graduating, Jenna plans to continue working in the area of skeletal

muscle research.





PAGE 1

1 REDUCED SENSITIVITY TO NITRIC OXI DE LIMITS SATELLITE CELL ACTIVITY ON CULTURED MYOFIBERS FROM AGED MICE By JENNA LEIGH JONES BETTERS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

PAGE 2

2 2007 Jenna Leigh Jones Betters

PAGE 3

3 To my husband, Chad Betters, who never doubt ed my capabilities, even when I did

PAGE 4

4 ACKNOWLEDGMENTS This work was completed with the help of ma ny people. First, I thank Dr. David Criswell, my supervisory committee chair. I gained valuab le knowledge during my 6 years working in his lab. He guided me through the completion of this often-daunting degree. I thank my committee members (Drs. Scott Powers and Steve Dodd from Applied Physiology and Kinesiology, and Dr. Sally J ohnson from Animal Sciences). Each has contributed valuable insight to this pr oject, and to my graduate experience. Dr. Grace Pavlath and Brenda Bondesen from Em ory University graciously taught me the fiber isolation technique. I greatly appreciate their expertise and willingness to assist me. The members of the Molecular Physiology lab deserve some credit, too. Quinlyn Soltow, Vitor Lira, and Jeff Sellman, have been my fe llow lab rats throughout my graduate student tenure. Vitor and Dana Brown deserve extra credit for all the time spent on the Dark Side to complete the DAF experiments. Lastly, I would not be in gra duate school and completing this dissertation wit hout the love and support of my family. My husband, Chad Bette rs, and my parents and sister, Jim and Sue Jones and Julie Jones, provided the behind the s cenes support that made this work possible.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ..............9 CHAPTER 1 INTRODUCTION..................................................................................................................11 Background..................................................................................................................... ........12 Problem Statement.............................................................................................................. ....13 Variables in Study............................................................................................................. ......14 Specific Aims and Hypotheses...............................................................................................14 Limitations/Delimitations/Assumptions.................................................................................16 Significance of the Study...................................................................................................... ..16 2 LITERATURE REVIEW.......................................................................................................17 Satellite Cell Activation...................................................................................................... ....17 Hepatocyte Growth Factor (HGF)...................................................................................18 Nitric Oxide (NO)............................................................................................................18 Stretch........................................................................................................................ ......20 Recovery from Atrophy...................................................................................................21 Aging and Muscle Satellite Cell Activity...............................................................................23 Satellite Cell Numbers with Age.....................................................................................23 Aging and Muscle Regeneration.....................................................................................25 Notch Signaling and Satellite Cell Activation.................................................................27 Nitric Oxide and HGF/c-Met in Aged Muscle................................................................30 Summary........................................................................................................................ .........31 3 MATERIALS AND METHODS...........................................................................................33 Experimental Designs........................................................................................................... ..33 Animals........................................................................................................................ ...........34 In Vitro Electrical Stimulation and Measur ement of Nitric Oxide Production......................34 Fiber Isolation................................................................................................................ .........36 Immunohistochemistry...........................................................................................................37 Asymmetric Dimethylar ginine (ADMA) ELISA...................................................................38 Western Blotting............................................................................................................... ......38 Statistical Analysis........................................................................................................... .......39

PAGE 6

6 4 RESULTS........................................................................................................................ .......40 Muscle Atrophy in Male C57 Mice........................................................................................40 Nitric Oxide Production fr om Contracting Muscle................................................................40 Nitric Oxide Synthase Protei n Expression Is Not Reduced....................................................41 Asymmetric Dimethylarginine Can Inhibit Satellite Cell Activity........................................41 Asymmetric Dimethylarginine Is Not Elevated in Aged Muscle and Blood.........................42 Satellite Cell Response to HGF Is Not Altered with Age......................................................42 L-Arginine and a Nitric Ox ide Donor Can Rescue Aged Satellite Cell Activity...................42 5 DISCUSSION..................................................................................................................... ....56 Main Findings.................................................................................................................. .......56 Reduced Satellite Cell Activation in Response to Mechanical Perturbation..........................56 L-Arginine and a Nitric Ox ide Donor Can Rescue Aged Satellite Cell Activity...................57 Nitric Oxide Production and NOS Prot ein Expression from Aged Muscle...........................58 Satellite Cell Response to HGF Is Not Altered with Age......................................................60 Asymmetric Dimethylarginine Is Not Elevat ed in Aged Muscle and Blood but Does Inhibit Satellite Cell Activity..............................................................................................60 Response to Nitric Oxide....................................................................................................... .62 Limitations and Future Directions..........................................................................................63 Conclusions.................................................................................................................... .........64 LIST OF REFERENCES............................................................................................................. ..67 BIOGRAPHICAL SKETCH.........................................................................................................72

PAGE 7

7 LIST OF TABLES Table page 4-1 Body mass, gastrocnemius mass, muscle mass/body mass ratios, and total protein for young, adult, and old mice.................................................................................................44 4-2 Asymmetric dimethylargi nine (ADMA) concetration ( mol/L) in serum and muscle of young and old mice........................................................................................................45 4-3 Quantification of satellite cell activati on as measured by (A) cell emanation and (B) BrdU incorporation from young, adult, and old myofibers...............................................46

PAGE 8

8 LIST OF FIGURES Figure page 4-1 Nitric oxide (NO) pr oduction as quantified by 4,5diaminofluorescein (DAF) fluorescence................................................................................................................... ....47 4-2 Representative immunoblot of neuronal ni tric oxide synthase from young, adult, and old plantaris muscle...........................................................................................................48 4-3 Representative immunoblot of endothelial nitric oxide synthase from young, adult, and old plantaris muscle.....................................................................................................49 4-4 Representative images of an emanating satellite cell and a BrdU+ nucleus......................50 4-5 Satellite cell emanation and BrdU inco rporation of fibers following exposure to ADMA........................................................................................................................... ....51 4-6 Representative image of a fiber stained for pax7 immedi ately following isolation and plating. ...................................................................................................................... ........52 4-7 Satellite cell emanation and BrdU inco rporation of fibers following exposure to HGF............................................................................................................................ ........53 4-8 Satellite cell emanation and BrdU inco rporation of fibers following exposure to Larginine and DETA-NO. ...................................................................................................54 5-1 Overview of the steps lead ing to satellite cell activation and the potential location of the aging deficit.............................................................................................................. ....66

PAGE 9

9 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy REDUCED SENSITIVITY TO NITRIC OXI DE LIMITS SATELLITE CELL ACTIVITY ON CULTURED MYOFIBERS FROM AGED MICE By Jenna Leigh Jones Betters August 2007 Chair: David Criswell Major: Health and Human Performance Although aging-induced deficits in satellite cell pr oliferation and musc le regeneration are well-characterized, our investigatio n provides novel evidence of an age-related deficit in satellite cell activation, independent of prol iferation, that is intrinsic to the muscle fiber. Mechanical activation of muscle fibers induces enzymatic production of nitric oxide (NO) resulting in proteolytic release of hepatocy te growth factor (HGF) from its extracellular tethering, and subsequent activation of satellite cells. We hypothesized that aging would reduce in situ satellite cell activation, and that L-argini ne or nitric oxide (DETA-NO) supplementation would improve satellite cell activity in old musc le. Single intact myofibers were isolated from the gastrocnemius muscles of young (2 mo), adult (10 mo), and aged (22 mo) male C57 mice. Fibers were centrifuged to stimulate satellite cell activati on and incubated in serum-free media with 0.002% 5-bromodeoxyuridine (BrdU) and either 2 mM L-arginine, 10 M DETA-NO, or control media for 48 h. The total number of satellite cells per fiber (i.e., pax7+) was unchanged with aging. Nevertheless, the number of ac tivated satellite cells (BrdU+) following centrifugation was reduced in the aged fibers. L-argi nine or DETA-NO treatment to the aged fibers restored satellite cell activation to young-control leve ls. However, these treatments also increased satellite cell activation in young and adult fibers, such that an age-dependent de ficit in satelli te cell activity

PAGE 10

10 persisted within treatment groups. Expression of neuronal and endothelial nitric oxide synthase (NOS) proteins in the plantaris muscles did no t differ between age groups. Further, contractioninduced NO release from the extensor digitoru m longus (EDL) did not differ between adult and old mice. These data suggest that impairme nt of NO production does not account for reduced satellite cell activity in aged muscle fibers. Also, treatment with exogenous HGF was equally effective in activating satellite cells across age groups, indi cating that events downstream of HGF release are intact in the aged muscle. Therefore, these data imply a reduced sensitivity of aged muscle to NO. Insight gained from these ex periments will augment therapeutic strategies to maintain skeletal muscle mass in the elderly, and to enhance muscle regeneration in this population following disuse or acute injury.

PAGE 11

11 CHAPTER 1 INTRODUCTION Sarcopenia, the gradual declin e of skeletal muscle mass a nd strength, is a hallmark of aging. Sarcopenia is generally responsible for the age-related decline in physical performance, and ultimately for physical disability in many elderly people. The prevalence of sarcopenia, defined as appendicular skel etal muscle mass (kg)/height2 (m2) being less than two standard deviations below the mean of a young reference group, ranges from 13 to 24% in those 65 to 70 years old, and is over 50% for t hose greater than 80 years of age (6). Another associated characteristic of aging is a declin e in ability of skeletal muscle to repair itself following damage. Age-associated muscle atrophy likely results, in part, from the reduced ability of muscle to regenerate following repeated micro-trauma occu rring during the course of daily activities (e.g., eccentric contraction-induced injury). Mature skeletal muscle fibers consist of postmitotic and terminally differentiated nuclei. Thus, the addition of nuclei to skeletal muscle fibers during hypertrophy or regeneration must come from sources other than m yonuclei. Satellite cells represent the muscle stem cells that provide these additional nuclei. Satellite cells are quiescent precursor cells residing in a satellite position alongside muscle fibers (37). The ability of these muscle precursor cells to increase muscle mass and repair muscle following injury makes them essential to skeletal muscle function. However, with advanced age, there is a reduction in the population of satellite cells (5, 12, 28, 31, 45, 48, 51) and their ability to activate/prol iferate, and repair muscle (19, 20). Thus, the ability of aged muscle to increase mass or re pair damage is poor compared to young or adult muscle. The mechanisms underlying the age-related defi cit in satellite ce ll function warrant exploration.

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12 Background Muscle atrophy during aging likely results, in part, from inefficient muscle repair consequent to injury. Skeletal muscle regene ration following immobilization-induced atrophy is lacking in aged rats, even after 77 days of recovery (17). Howeve r, direct administration of IGF1 onto atrophied muscles restores the in vivo regenerative capacity of old muscle and in vitro satellite cell proliferation. This finding suggests that satellite cells from aged muscle do not lose their inherent ability to proliferate, but simp ly are not activated sufficiently to regenerate following an injury. Thus, it is important to isol ate the process of activ ation from subsequent proliferation. Old satellite cells may remain in active due to the lack of some unknown growth factor/mitogen, or the failure to respond to the levels of endogenous growth factors. Satellite cells are known to be activated by several stimu li, such as hepatocyte growth factor (HGF) (4, 53, 54), mechani cal stretch (54, 59) and exerci se (23, 24, 32). Activation of satellite cells refers to entr y into the cell cycle from quiescen ce, including DNA synthesis, and mobilization. Other growth factors, such as IGF1 and basic FGF, can stimulate proliferation of satellite cells (7, 8, 9, 10, 51, 61) but t hus far, only HGF affects activation. Nitric oxide (NO) appears to be an important signaling molecule mediating satellite cell activation via HGF release (4). Inhibition of NO production inhi bits HGF release, c-met/HGF co-localization, and satel lite cell activation (2). In addition, mechanical stretch of rat satellite cells (54) and isolated mouse myofibers (59) id entified the importance of NO in stretch-induced satellite cell activation. Similarly, HGF is crucial for stretch-indu ced satellite cell activation, and its release is dependent on NO (54). Satellite cell proliferation and cell fate de termination are regulated by the Notch signaling pathway. This pathway is triggered by the rapid upregulation of Delta, the Notch ligand, following injury. Recently, Conboy et al. (19) demons trated deficiencies in Notch signaling that

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13 affected proliferation and regene ration of old mouse muscle. They showed that old muscle fails to upregulate Delta in response to an injury. Fo rced activation of Notch improved regeneration in old muscle, while Notch inhibition decreased repa ir in young muscle. When old cultured satellite cells were exposed to young serum, satellite cells upregulated Delta and ac tivated Notch (20). In addition, parabiotic pairing of a young and an old mouse, such that they shared a circulatory system, restored regenerative potential followi ng an injury in the old mouse muscle. These experiments demonstrate that Notch activation is necessary for regeneration in young muscle and sufficient to stimulate effec tive repair in old muscle. Pl us, components in young serum are capable of reversing the age-induced decline in ac tivation/proliferation of satellite cells. Thus, satellite cells do not lose their i nnate ability to repair muscle wh en aged, but modifications of the old systemic environment pr event their proper functioning. Problem Statement A better understanding of sarcopenia is esse ntial since a significant proportion of the population is living long enough that muscle atrophy becomes a major issue. In addition, physical rehabilitation following an injury requires functional satell ite cells. Aging is associated with an impaired ability to repair muscle fo llowing damage, and an increased amount of muscle wasting. Advanced age reduces satellite cell numbers (5, 12, 28, 31, 45, 48, 51) and satellite cell activation/proliferation (19, 20) Work by Conboy and colleagues (20) has shown that aged satellite cells can repair muscle when exposed to a young systemic environment. This suggests that extrinsic signals regulate aged satellite cell activity, and that the possi bility exists that the loss of muscle regenerative capacity can be restored if the satelli te cell environment more closely resembles that of a young animal. Given the im portance of NO as the initiating signal for satellite cell activation, we pr opose that one key age-related modification in the muscle environment is a decrease in NO bioavailabilit y. We postulate that NO production is reduced

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14 with aging and may compromise satellite cell function. NO producti on could be reduced due to a decrease in the cationic amino acid transporter (CAT) expression responsible for transporting the NOS substrate L-arginine into the cell, a re duction in NOS expressi on, or an increase in asymmetric dimethylarginine (ADMA), an e ndogenously produced metabolite which inhibits NOS activity. Discovery of the age-associated factor s that regulate satellite cell behavior will be instrumental for designing therapies targeted to augment the regenerative potential of muscle and thus maintain muscle mass in elderly individuals. Variables in Study Independent variables: We will manipulate the ag e of the animals, electrical stimulation of incubated muscles, and the isolated myofib ers treatment medium (supplementing with Larginine, DETA-NO, HGF, or ADMA). Dependent variables: We will measure satellite cell activation via BrdU incorporation. We will also measure nNOS, eNOS, and iNOS protein content in plantaris muscle, CAT1/2 mRNA levels, and stain for Pax7 and BrdU in isolat ed myofibers. We will measure nitric oxide production in extensor digitorum longus (E DL) muscles electri cally stimulated in vitro Control variables: Only male C57 mice will be studied, so gender is pu rposely excluded from this study. Extraneous variables: We will not control prior activity level or food and water intake of the mice. Specific Aims and Hypotheses This study is designed to an swer the following questions: Question 1. Is nitric oxide produc tion reduced in old muscle?

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15 Hypothesis 1: NO production (qua ntified by DAF fluorescence) is reduced in old muscle following in vitro electrical stimulation of the EDL. In addition, NOS protein expression and CAT mRNA are reduced in old muscle. Question 2. Is there a deficit in satellite cell activa tion with aging in response to exogenous HGF? Hypothesis 2. Aged satellite ce lls will respond to increasing doses of HGF by incorporating BrdU into DNA similar to young satellite cells, in dicating that the deficit is upstream of HGF release. Question 3. Is asymmetric dimet hylarginine (ADMA) elevated in the muscle and/or blood of aged animals? Hypothesis 3: ADMA, as measured by a commercia lly available ELISA kit, is elevated in both the serum and muscle of aged animals. Question 4. Can ADMA inhibit satellit e cell activation of young fibers? Hypothesis 4: Physiologically relevant concentrations of ADMA will reduce satellite cell activation of young fibers in response to mechanical perturbation (centrifugation). Question 5a. Is there a deficit in satellite cell activation with aging in response to mechanical stress? Hypothesis 5a: Aged satellite cells will have de creased activation (cell emanation as well as BrdU incorporation) of satellit e cells in response to mechanical perturbation (centrifugation). Question 5b. Can L-arginine and/or a nitric oxide donor (DETA-NO) rescue satellite cell activation in old muscle? Hypothesis 5b: Both L-arginine and DETA-NO treatment for 48 hours will return satellite cell activation of old fibers to young levels.

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16 Limitations/Delimitations/Assumptions Limitations: The invasive nature of this st udy negates the use of human subjects. A mouse hindlimb model has been selected because of the similarities in structur e and function of mouse and human skeletal muscle. Delimitations: Gender and species differences may ex ist in regard to sate llite cell function. We have chosen to study only male C57 mice. Assumptions: It is assumed that satellite cell s remain quiescent during the myofiber isolation procedure. Previous experiments have confirme d the quiescent nature of satellite cells during isolation using this technique. Significance of the Study Normal daily activity subjects skeletal muscle to injuries, necessitating a means of repair to maintain muscle. Thus, there is a constant demand for satellite cells thro ughout life. A decrease in satellite cell abundance and activity with age may contribute to the ageassociated deficiency in muscle repair, and ultimately to muscle loss. This research will improve our knowledge of the mechanisms underlying the deficit in satellite cell function with aging. We seek to identify aging-asso ciated deficiencies in signaling pathways leading to satellite ce ll activation, and to reverse these deficiencies. This study will provide insight into clinical th erapies to attenuate muscle loss and improve muscle regeneration in the elderly.

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17 CHAPTER 2 LITERATURE REVIEW Skeletal muscle is a multi-nucleated tissue capab le of generating force that is essential for locomotion and breathing. It is continually subjec ted to injury and micro-trauma, resulting from mechanical stress, including we ight bearing and exercise. Thus skeletal muscle requires a renewable source of cells for muscle repair. Satel lite cells provide the additional nuclei to muscle fibers in times of growth or regeneration. Satellite cells are located adj acent to the muscle fiber and beneath the basal lamina (37). They are believed to be the committed stem cells of adult skeletal muscle (57). While normally quiescent, they are activated when needed fo r repair or to mediate muscle hypertrophy. Aging is associated with a decreased sate llite cell number (5, 12, 28, 31, 45, 48, 51) and marked impairments in regeneration following in jury or atrophy (17, 19, 20, 43). This project seeks to understand the molecular mechanisms of satellite cell aging that lead to loss of tissue regenerative capacity in old muscle. Satellite Cell Activation Satellite cell activation is defined as entry into G1 phase from quiescence, and mobilization (2). To date, the only known factors to activate sa tellite cells include hepa tocyte growth factor (HGF) and nitric oxide (NO). Perturbations such as stretch and exerci se act via NO and HGF release to activate satell ite cells. In addition, th e activating factor in crushed muscle extract (CME), a known activating treatment, is HGF (53) Other growth factors, such as insulin-like growth factor-1 (IGF-1) and ba sic fibroblast growth factor (b FGF), cannot directly activate satellite cells, but do improve proliferation (7, 8, 9, 10, 61).

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18 Hepatocyte Growth Factor (HGF) HGF is synthesized in an inactive form as a si ngle chain of ~90 kDa, and is proteolytically cleaved into an active, disulf ide-linked heterodimer of 60 kDa (alpha-chain) and 30 kDa (betachain) by serine proteases (40, 41) Tatsumi and Allen (52) showed that HGF is present in the extracellular domain of uninjur ed rat skeletal muscle as act ive heterodimeric HGF. Significant amounts of pro-HGF were only found in rat muscle when the integrity of the membrane was compromised. Thus, active HGF is present in the ex tracellular matrix (ECM) and can be released following injury. This is important because prot eolytic conversion of pro-HGF to active HGF is therefore not a rate-limiting step in th e release of active HGF from the ECM. HGF is released from its tethering to the ECM by the action of matrix metalloproteinases (MMPs) (62). When released, HGF rapidly co-local izes with its receptor on satellite cells, the cmet tyrosine kinase receptor and triggers activation from quiescence (53). Once activated, satellite cells synthesize both HGF and c-met (3, 53). Treating muscle with CME causes dose-depende nt activation of sate llite cells (11, 53). CME increases expression of pr oliferating cell nuclear antige n (PCNA), an indicator of activation (DNA synthesis), in th ree week-old and nine month-ol d rat satellite cells (30). The factor in CME causing activation was later identifi ed as HGF (53). Treating satellite cells with CME in the presence of anti-HGF neutraliz ing antibodies eliminated the CME-induced proliferation, indicating that HGF is the factor in CME responsib le for stimulating satellite cell activity. Therefore, HGF is an important growth fact or to skeletal muscle because of its role in activating satellite cells and thereby c ontributing to repair and regeneration. Nitric Oxide (NO) Work has shown that HGF release from th e extracellular matrix is NO-dependent, and inhibition of nitric oxide synt hase (NOS) prevents that rel ease (2, 4, 50, 52). Anderson and

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19 colleagues have demonstrated that NO synthesis is implicated in the activation of satellite cells in crush injured skeletal muscle (2 ) and resident satellite cells on isolated myofibers cultured in vitro (4). In vivo NOS inhibition prevents HGF/c-met colocalization and satellite cell activation (2). Activation of satell ite cells on single myofibers was found to be dependent on the levels of NO (related to the amount of substr ate, L-arginine), and HGF in th e media (4). In contrast, NOS inhibition (L-NAME) prevented this activation. Anderson and Pilipowicz (4) char acterized the series of even ts leading to satellite cell activation on isolated single myofibers. CME, HGF, and L-arginine activated satellite cells, while L-NAME significantly blocked the potent activation by CME, as well as spontaneous control activation. These results suggest that NO plays a role in spontaneous and HGF-dependent activation. Since spontaneous activation was decr eased by NOS inhibition but restored by HGF, the activation signaling by HGF appears to o ccur after NO-mediated signaling events. Tatsumi and Allen (52) furthered our unde rstanding of NO-mediated HGF release by showing that the NO donor, sodium nitroprusside (SNP, 300 M), caused release of active HGF from the extracellular matrix of rat EDL muscle and did not involve proteolytic cleavage of proHGF. The addition of protease inhibitors to the incubation medium did not increase the amount of pro-HGF released, demonstrat ing that the NO-stimulated m echanism of active HGF release does not require proteolytic cleavage of pro-HGF In addition, Yamada and colleagues (62) provided evidence of the role of MMPs in the NO-dependent release of HGF from the extracellular matrix. More recently, Wozniak and Anderson (58) dem onstrated that NO concentration regulates the balance between satellite cell quiescence an d activation on isolated fibers. A normal concentration of NO maintains qu iescence, while higher or lower levels of NO induce activation.

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20 This was shown by stretching normal fibers and increasing NO concentration and satellite cell activation. Likewise, satelli te cell activation was increased in models of reduced NO availability, namely fibers from mdx mice and NOS-I -/mice. These experiments hi ghlight the importance of NO for satellite cell activity. Stretch Mechanical stretch of cultured adult rat satelli te cells was shown to cause release of HGF and accelerated entry into the cell cycle (54). Conditioned media from cultures of stretched satellite cells stimulated activation of unstretched cultures, and anti-HGF neutralizing antibodies in conditioned media eliminated this respons e. Additionally, conditioned media from cells stretched with L-NAME did not cau se activation of unstretched sa tellite cell cultures, and HGF was not released into the media. Media from uns tretched cells cultured in the presence of an NO donor was able to increase activation of control cu ltures, and HGF was det ected in this media. NOS activity assays indicated that NOS was s timulated when muscle satellite cells were stretched. This data indicates th at stretch stimulates intracellu lar events involving NO synthesis and subsequent HGF release lead ing to satellite cell activation. Mechanical stretch of single fibers has been us ed to activate satellite cells associated with myofibers (59). Satellite cells on stretched fibers were signifi cantly activated (incorporated BrdU) at 30 minutes and 2 hours after initiation of stretch. It was speculated that these two peaks may indicate that the same sate llite cells entered pro liferation after 30 mi nutes and 2 hours of stretch and that each cell requires more than one stimulus to activate. Or, that the two peaks symbolize two or more populations of satellite cells entering pro liferation after differing stretch stimuli (30 minutes and 2 hours). The latter explan ation suggests that diff erent regulatory events may exist among various subpopulat ions of satellite cells.

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21 This same study (59) also examined c-met expression in control, quiescent fibers. The number of quiescent c-met+ cells/fiber, determined using in situ hybridization with c-met mRNA, was found to be approximately 1.4, with a range of 0-6 per fiber, in non-stretched cultures. There are a number of c-met+ cells that are not activated (BrdUnegative) by an activating stimulus (HGF, CME, or stretch), indicati ng that the entire popul ation of satellite cel ls is not uniformly activated. This may be due to a subset of satell ite cells that remain quiescent to maintain the precursor pool. The use of stretc h of single myofibers has the poten tial to further elucidate the mechanisms of stretch-induced act ivation of satellite cells in th eir resident position and from a quiescent state. Mechanical stretch activates sa tellite cells on fibers (59) a nd in single cell cultures (54). NOS activity is stimulated by stretch, and activ ation is NO-dependent (5 4). Likewise, HGF is critical for activation and its stretch-induced rele ase is prevented with NOS inhibition. Therefore, NO appears to be an important signal for st retch-induced HGF release and satellite cell activation. Recovery from Atrophy Skeletal muscle atrophies greatly in re sponse to unloading. Two weeks of hindlimb suspension resulted in a 36% reduction in myonu clear number in the mo use soleus (38). Two weeks of recovery restored myonuclear number and muscle mass, suggesting that muscle precursor cells can proliferate and fuse with myofibers. However, local gamma-irradiation, which inhibits muscle precursor cell proliferatio n, prevented the recovery in muscle mass and myonuclear number following unloa ding. Growth was normal in the first week of recovery, but inhibited in the second week. These data indicate that proliferating muscle precursor cells are required to completely restore myonucle ar number and muscle mass after atrophy.

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22 This same group showed that hindlimb susp ension of two weeks reduced (by 14%) the total number of cells isolated from atrophied muscle s, and the number of sa tellite cells associated with isolated myofibers compared to cont rol (39). Atrophied myofibers produced fewer myoD+/desmin+ cells per myofiber compared to nonatrophied fibers. To assess whether proliferation was affected by hindlimb suspensio n, isolated precursor cells were labeled with BrdU. A 25% decrease in BrdU+/desmin+ cells was found in atrophie d muscles, suggesting the ability to activate and/or prolif erate was impaired. The ability of isolated cells from atrophied muscles to differentiate and form myotubes was adversely affected by hindlimb suspension, suggesting that differentiation a nd fusion were also affected. C onversely, recovery from atrophy (normal ambulation) reversed all the effects of atr ophy. Total cell number and myoD+/desmin+ cells per myofiber were increased. More BrdU+/desmin+ cells were associated with recovered muscles, and formation of large myotubes from mu scle precursor cells was restored. Therefore, no permanent changes occur in the muscle precursor pool with at rophic conditions. Upon cessation of the atrophic stimulus all changes were reversible. Similarly, casting muscle for extended peri ods of time and then allowing for recovery causes muscle wasting followed by muscle repair. Although a decline in sate llite cell number is evident with immobilization in the adult rat, remo bilization leads to complete recovery of muscle mass and myofiber regeneration (13). While prot ein synthesis increases during the recovery phase, muscle precursor cell proliferation also increases to provide a dditional nuclei (35). The satellite cells of adult animals are therefore able to activate and prolifer ate to promote recovery of atrophic muscle. Skeletal muscle has the unique ability to repair and regrow, or to hypertrophy with appropriate stimuli. However, these actions can not occur without the addition of new nuclei to

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23 existing myofibers, or the fusion of myoblasts to form new myofibers. The source of these new nuclei is the muscle precursor pool of satellite cells. Satellite cell activation is therefore an indispensable step in muscle repair and adaptatio ns to increased load. To date, HGF is the only growth factor able to directly activate satelli te cells, and its release is dependent on NO. Aging and Muscle Satellite Cell Activity Sarcopenia is the loss of skeletal muscle mass and strength with age. The age-related muscle atrophy has been attributed to marked sa tellite cell quiescence (25). In aged muscle, fewer satellite cells are present (5, 12, 28, 31, 45, 48, 51) and they exhibit a long latent period between the activating stimulus and proliferation (53, 61). However, data suggest that aged satellite cells retain their intrinsic myogenic pot ential, but the environmental stimuli are altered (20, 51). Satellite Cell Numbers with Age Animal studies indicate that the satellite cell population decl ines with aging (5, 12, 28, 51). However, results from human skeletal muscle are less harmonious. Many researchers report a reduced proportion of human satellite cells wi th aging (31, 45, 48), while others report no changes compared to young muscle (29, 47). Renault et al. (45) found the proportion of satellite cells in the biceps brachii and masseter muscles was significantly lower in elderly men (mean age, 74 years-old) compared to younger men (mean age, 23 years-old). Kadi et al. (31) studied moderately active men and women, and found the elderly (age range, 70-83 years-old) had a significantly lower number of sate llite cells and proportion of satellite cells compared to young subjects (age range, 20-32 years-ol d). Also, Sajko et al. (48) analyzed autopsy samples of vastus lateralis muscle from young (mean age, 28.7 year s-old) and old (mean age, 70.8 years-old) and determined that numbers of satellite cells per fi ber length and proportion of satellite cells were

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24 significantly lower in the old muscles. These studies involving huma n subjects support the hypothesis that the satellite cell fraction declines with age. In contrast, Hikida et al. (29) sampled young (mean age, 22.5 years-old) a nd elderly (mean age, 65 years-old) men before and after a pe riod of strength traini ng, and found no significant differences in the proportion of satellite cells be tween the age groups. Similarly, Roth et al. (47) found no significant differences between the satell ite cell population of young (20-30 years-old) and old (65-75 years-old) men and women. Seve ral factors may contribute to the opposing results reported by various researchers. First, th e number of individuals sampled and their ages may affect the results. In general, age-related ch anges are clearly visible af ter age 70 (34). In the study by Renault et al. (45), the mean age was 74 a nd 4 of the 6 subjects were 78 years of age or over. In contrast, the mean age of the elderly group was only 65 years in the study by Hikida et al. (29). Other variables that ma y affect the differing results in clude different muscles sampled, and the training status of individuals. Most animal studies report declines in satellite cell numbers with age (5, 12, 28, 51). Muscle-derived cell yield was si gnificantly reduced in old rat mu scle compared to young (5). Bockhold and colleagues (12) reported a decline in the number of myogenic cells with aging in the mouse using cells that migrated from single fi bers in culture. Shefer and colleagues (51) used molecular markers to characterize quiescent satellite cells and their progeny as they proliferate, differentiate, and replenish the re serve pool in mouse muscle. The re sults of this study (51) show that resident satellite cell numb ers decline with age in myofibers from both fastand slow-twitch muscles. Enrichment of the cult ure medium with FGF2 enhances the regenerative capacity of aged satellite cells, indicating th e myogenic potential of aged satelli te cells is not decreased as abundance declines.

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25 In contrast, Yablonka-Reuveni et al. (61) found that myofibers cultured from old rat muscle retained a greater number of proliferating myoD+ cells than young rat muscle. However, the ages of old rats in this study were 9-11 months-old, and were compared to young (3 weeksold) rats in the rapidly grow ing phase. Conflicting data from various groups may result from differences in species used, ages sampled, and techniques to identify satellite cells. Aging and Muscle Regeneration Following limb immobilization, young muscle has th e ability to complete ly regain muscle mass (13). In contrast, old muscle exhibits remarkably little regrowth following limb immobilization (17, 43). The gastrocnemius of old rats showed no significant muscle regrowth, even after 77 days of recover y. In addition, the soleus muscle of old rats fails to show any regrowth after 30 days of recovery (43), wher eas the soleus of young muscle recovers 37 and 40% of mass after 6 and 15 days of reload, resp ectively, after the same atrophy stimulus (18). Chakravarthy et al. (17) admi nistered insulin-like growth fa ctor-1 (IGF-1) directly onto atrophied muscle of 25-30 month-old rats, and found that in vivo skeletal muscle regrowth and in vitro satellite cell proliferation were stimulated. They further showed that IGF-1 improves proliferation by decreasing the cell cycle inhibi tor p27Kip1 via the PI3kin ase/Akt pathway (16). These data prompted the hypothesis that satellite cells were in su fficient quantity in old muscle, but the systemic environment lacked appropriate growth factors/stimu li, or that old satellite cells were less responsive to stimuli. Age-related muscle loss could pa rtially result from repeated episodes of incomplete repair and regeneration (5). Muscle-derived cells (mdc ) from young rats started to proliferate sooner than adult and aged mdc. Cell cycle duration wa s faster in old cells compared to young, which could lead to a more rapid exhaustion of replic ative potential. Serum stimulation significantly induced c-met and PCNA expression in young mdc, but the increase was much lower in old mdc.

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26 When measured on crushed muscle extracts plasminogen activators (PAs) and matrix metalloproteinase 2-9 (MMP 2-9) enzyme activities were significantly lower with aging. These modifications in events associated with satell ite cell activation and proliferation may lead to repeated bouts of incomplete repa ir and regeneration and to the lo ss of skeletal muscle mass with aging. The hypothesis of incomplete repair by Barani et al. (5) is supporte d by others (26, 27). Gallegly and colleagues (27) f ound evidence of ongoing regeneration in old rat muscle. Centrally located nuclei were elevated in the soleus of aged (32 month-old) compared to young (6 monthold) and adult (20 month-old) ra ts. Myonuclear domain size was d ecreased with age since soleus muscle atrophy was not accompanied by a change in myonuclear number. Total BrdU+ nuclei were decreased with age, but BrdU+ myonuclei were increased. These results suggest that muscle atrophy with age is coincident wi th increased myofiber nuclear density from proliferation and fusion of satellite cells, resembling ongoing rege neration. Likewise, Edstrom and Ulfhake (26) found no lack of regenerative drive in aged rat mu scle. Aged soleus muscle had an accumulation of nuclei, a wide distribution of fiber size s, and an increase in embryonic MHC staining compared to young muscle. The gastrocnemius mu scle had high levels of MyoD and myogenin mRNA expression compared to young. The regenera tive drive is apparently intact in aged muscle, but the efficiency or outcome of regeneration is poor. In young rats, intermittent reloading for one hour/day during 7 or 14 days of hindlimb suspension was sufficient to attenuate soleus musc le atrophy (27). However, this intervention did not attenuate atrophy in aged (32 month-old) ra ts. Intermittent reloading was only associated with an increase in BrdU+ nuclei in young rats. This altered res ponse of the soleus to an atrophyreducing stimulus likely results from alte red satellite cell ac tivation with age.

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27 Conflicting data concerning myonuclear domain were published by Brack et al. (14). This group found that loss of nuclei per unit of fiber lengt h with age is exacerbated in larger fibers, resulting in an increased domain size in these fibers, whereas Gallegly et al. (27) reported a decrease in nuclear domain size. This loss of nuc lei reported by Brack and colleagues (14) can be accounted for by either excessive loss, or insufficient replacement. These researchers hypothesize that muscle atrophy occurs to restore the nuclear domain size of a fiber to its setpoint after a transient increase in domain size due to nuclear loss /inadequate replacement. Thus, the debate seems to be whether nuclei are lost prior to fiber atrophy, or vice versa. Gene regulation during recovery from atrophy is altered in aged muscle (1, 27, 36, 43). In particular, MyoD remained elevated in old musc le after regeneration, wh ereas its levels were restored to normal in young animals (36). Plus aging reduced the incr ease in MyoD protein levels during hindlimb suspension (1). With adva ncing age, the increase in MyoD and myogenin mRNA with hindlimb suspension in young rats was ab sent (27). Therefore, it is highly probable that genes regulated by these tr anscription factors are dysregula ted. Pattison et al. (43) also demonstrated altered gene regul ation with recovery from atrophy in aged muscle. A growth factor (amphiregulin) was not upregulated, while a cell death-associated protein (clusterin) remained upregulated in the old ve rsus the young rat soleus muscle. Notch Signaling and Satellite Cell Activation Conboy and Rando (21) utilized an isolated m yofiber model to study the role of Notch-1 and its antagonist Numb in satellite cell activat ion. Muscle injury (tibialis anterior) led to increased expression of activated Notch-1 in myofibers isolated 72 hours after injury. Interestingly, Numb immunosta ining indicated an asymmetric distribution in myofiberassociated mononucleated cells. Numb and pax3, a premyoblast marker, were virtually mutually exclusive, whereas Numb mostly co-expressed with myf-5, pax7, and desmin. Therefore, Numb-

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28 cells have a less committed muscle precursor phenotype, while Numb+ cells have a more committed myogenic phenotype. Expression of constitutively active Notch-1 in muscle progenitor cells increased pax3 expression and decreased myf-5, myoD, and desmin (21). Constitutively active Notch also enhanced myoblast proliferation while re ducing embryonic myosin heavy chain (MHC) expression and the production of multinucleated myotubes. In contrast, overexpressing Numb decreased pax3 expression and increased myf-5 and desmin protein levels. High levels of Numb led to myotube formation. Taken together, these data indicate that Notch-1 activity promotes myogenic cell proliferation, while inhibition of Notch-1 activity by Numb leads to exit from the cell cycle, expression of musc le regulatory factors (MRFs) and myogenic differentiation. The Notch-1 ligand, Delta, increases in response to muscle injury in sa tellite cells of young (2-3 month-old) and adult (5-7 month-old) mice, but fails to do so in sate llite cells of old (23-24 month-old) mice (19). Increased Delta expression in young and adult mice is coincident with decreased Numb expression and increased prolif eration. Satellite cells from old mice have similar Notch-1 levels, but consistently lowe r levels of activated Notch than young mice. Following muscle injury, Delta ex pression is upregulated in sate llite cells from young mice, but old satellite cells failed to do so. There was a pproximately one-fourth the number of activated satellite cells in old muscle as in young and adult after injury. When Jagged-Fc, an inhibitor of Notch activation, was introduced at injury site s, young muscle regenera tion was ineffective, similar to that seen in old muscle. Direct activation of Notch, using an antibody to its extracellular domain, markedly improved in vivo regeneration in old muscle, making it similar to young muscle. Therefore, Notch activation is not only necessary for young muscle regeneration but is also sufficient to induce muscle regeneration in old muscle.

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29 To further support these experiments, Conboy et al. (20) performed a series of parabiotic pairings between young (2-3 month-old) and ol d (19-26 month-old) mi ce. These parabiotic pairings allowed for a shared circulatory syst em, exposing old mice to factors present in young serum and vice versa. After 5 weeks of parabi osis, the hindlimb muscles were injured and injected with BrdU. Five days after injur y, young muscle of both isochronic (young-young) and heterochronic (young-old) pairings had regenera ted. In contrast, old muscle from isochronic pairings regenerated poorly. However, pair ing with a young mouse significantly enhanced muscle repair in old partners. This regenerati on was due almost exclus ively to activation of resident aged satellite cells and not to infiltration of cells from the young partner. Satellite cells from the aged partner in hetero chronic pairings showed marked Delta upregulation, whereas this induction was lacking in old isochronic pairi ngs. There was a slight inhibition of Delta upregulation in young satellite cells from hetero chronic pairings compared to young isochronic pairs. The researchers also studied the e ffects of heterochronic parabiosis in vitro (20). Exposure of satellite cells cultured from old muscle to serum collect ed from young mice improved Delta upregulation and Notch activation compared to ol d satellite cells cultured in old serum. Young serum also significantly increased myofiber-associat ed satellite cell prolifer ation of old cultures. Young satellite cells exposed to old mouse seru m exhibited reduced Delta upregulation. When Notch signaling was inhibited, proliferation was decreased in both young a nd old satellite cells cultured in young mouse serum. Taken together, these in vivo and in vitro data suggest that factors in old serum negatively influence satellite cell activation, proliferation, and muscle repair. However, components in young serum alone are suffi cient to restore satellite cell function in aged muscle. This implies that the old systemic environment is altered such that there is a

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30 decrease in a positive factor found in young serum, an increase in a negative or inhibitory factor, or both. Nitric Oxide and HGF/c-Met in Aged Muscle Neuronal nitric oxide synthase (nNOS) activ ity and percentage of NOS-containing fibers were significantly reduced in muscles from ag ed rats compared to young (46). In young (8 month-old) animals, nNOS ac tivity was highest in the EDL, which also had the greatest percentage of NOS-containing fibe rs. Activity was lowest in the soleus, and intermediate in the diaphragm. NOS activity was significantly reduced in all muscles of the 24 month-old animals, with EDL still having the greatest activity. In cont rast to these results, Capanni and colleagues (15) reported an increase in n NOS expression in 24 month-old rat gracilis muscle. However, this group measured NOS expression via immunobl ots and immunohistochemistry, whereas Richmonds and colleagues (46) measured func tional NOS activity. It is possible that NOS enzymatic activity is inhibited even with an upregulation of nNOS protein expression. Asymmetrical dimethylarginine (ADMA) is a likely candidate for the inhibition of NOS activity with aging. ADMA is an endogenous inhibitor of NOS, and is elevated in the serum of aged animals compared to young (60). The role of ADMA in endothelial dysfunction has been studied (22), and by blocki ng NO generation, ADMA promotes processes leading to atherogenesis, plaque progression, and plaque rupture. There are associations between elevated levels of ADMA and many cardiovas cular risk factors, which are also associated with advancing age. Xiong et al. (60) measured ADMA levels in the serum of young (6 month-old) and old (20 month-old) rats. ADMA le vels were significantly increased in the serum of the aged animals compared to the young. Serum L-arginine levels were similar between the two ages, so the Larginine/ADMA ratio was lower in old rats comp ared to young. Lastly, serum concentrations of nitrite were significantly decrease d in old animals. Thus, it may be that ADMA levels increase

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31 with age, and inhibit NO signaling in old musc le. To date, no studies have measured ADMA levels in young and old skeletal muscle. NO is important for triggering the release of HGF in the extracellular matrix to activate satellite cells (2, 4, 50, 52). Barani and colle agues (5) reported a lowe r increase in the HGF receptor, c-met, expression in cultured muscle-d erived cells (mdc) of old rats with serum stimulation compared to young. Young (3 week-o ld) and adult (9 month-old) mdc cultures increased relative c-met expression by ~190% with serum stimulation. In contrast, old (24 month-old) mdc increased c-met expression by just 130% with serum stimulation. Cell surface density of the c-met receptor was increased to the same extent in the three ages with serum exposure. In conclusion, muscle wasting associated with aging is well docume nted. Deficiencies in satellite cell activation/ proliferation, and incomplete repa ir of old muscle are also well recognized. Several groups have identified strategies to improve muscle regeneration in aged animals, including supplemental IGF-1 (17), fo rced Notch activation (19), and exposure to young serum (20). A decrease in c-met expression and nNOS activity, and an increase in the NOS inhibitor ADMA may contribute to the age-associated reduction in satellite ce ll activation. Summary Nitric oxide production tr iggering HGF release is an essen tial step in the activation of satellite cells from a quiescent state. With ag ing, nNOS activity is decreased (46) and serum levels of the NOS inhibitor ADMA are increased (60). It appears that aged satellite cells retain the intrinsic ability to activate and proliferate when e xposed to a young systemic environment (21). This young environment both exposes aged sa tellite cells to young sy stemic factors, and reduces their exposure to old systemic factors. Id entifying those factors that affect the activity of endogenous satellite cells will be crucial to improving the regenerative capacity of aged muscle.

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32 It is possible that if satellite cell activation and pr oliferation can be maintained with age, skeletal muscle loss due to sarcopenia will be prevented.

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33 CHAPTER 3 MATERIALS AND METHODS Experimental Designs This project was designed to answer the following questions with the accompanying experimental designs: Question 1 Is nitric oxide (NO) prod uction reduced in old muscle? Experiment 1 Extensor digitorum longus (EDL) muscle s from young, adult, and old mice were incubated in small tissue baths and NO productio n was measured using DAF fluorescence in the presence and absence of electrical stimulation. Question 2 Is there a deficit in satellite cell act ivation with aging in response to HGF? Experiment 2 Young, adult, and old single myofibers we re isolated from the gastrocnemius muscle and cultured with different concentrations of HGF (1-50 ng/ml) to activate satellite cells. Question 3 Is asymmetric dimethylargi nine (ADMA) elevated in the muscle and/or blood of aged animals? Experiment 3 Blood was collected and serum isolat ed from young, adult, and old mice for measurement of ADMA. Also, plantaris muscles from the three age groups were harvested and homogenized for measurement of ADMA. Question 4 Can ADMA inhibit satellite cel l activation of young fibers? Experiment 4 Gastrocnemius myofibers were isolat ed from young animals and exposed to different concentrations of ADMA (0.1-50 M). Question 5a Is there a deficit in sate llite cell activation with agi ng in response to mechanical stress? Question 5b Can L-arginine and/or a nitric oxide donor (DETA-NO) rescue satellite cell activation in old muscle?

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34 Experiments 5a and b Isolated gastrocnemius myofibers from young, adult and old mice (4 mice per age group) were mechanically stimulated by centrifugation and activ ated satellite cells were quantified by cell emanation as well as BrdU incorporation. Animals The subjects were young (~2 mo old, n=13), a dult (~10 mo old, n=8), and old (~22 mo old, n=6) male C57 mice. All were housed in the J. H illis Miller Animal Scie nce Center in specific pathogen-free housing. Animals consumed the same diet (mouse chow and water ad libitum), and were maintained on a 12 h light:12 h dark ph otoperiod. Mice were maintained in the facility for at least one week prior to experiments. C 57 mice are an accepted rodent model of aging and were selected since our lab and ot hers have successfully used them as subjects for single fiber and satellite cell isolation. All procedures were approved by the University of Floridas Animal Care and Use Committee. In Vitro Electrical Stimulation and Measurem ent of Nitric Oxide Production We validated a technique for quantifying the re lease of nitric oxide from isolated mouse skeletal muscles at rest and immediately follo wing contractions, using the cell impermeable fluorescent detector of nitric oxide, 4,5-diaminofluorescein (DAF-2, Cayman Chemical Company, Ann Harbor, MI). This method was first reported as a reliable method of quantifying NO release from cultured endothelial cells (33). Addition of the NO-donor, MAHMA-NO (0-100 nM), to oxygenated buffer containing DAF-2 ( 0.1 M) was used to establish the relationship between fluorescence intensity and NO concentration (R2=0.98, data not shown). The EDL muscle from one leg was electricall y stimulated for 10 mi n to quantify activityrelated nitric oxide production. Th e contra-lateral EDL was incubated without stimulation for the same amount of time. Animals were anesthetized with inhaled isoflura ne (2%) with oxygen as the carrier gas. Both EDL muscles were remove d and mounted in separate micro-tissue baths

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35 between two platinum electrodes positioned 8 mm apart and surrounding the central part of the muscle. Baseline tension was set for each muscle at 2.0 g of tension. During the experiment, muscles were incubated in oxygenated Krebs-He nseleit bicarbonate buffer (KHB buffer: 1.2 mM KH2PO4, 25 mM NaHCO3, 118.5 mM NaCl, 4.7 mM KCl, 0.25 mM CaCl2, 1.2 mM MgSO4, pH 7.4). The incubations were carried out at constant temperature (30oC) and under continuous gassing with 95% O2/5% CO2. After setting baseline tension (2.0 g), muscle s were incubated for 10 min. Muscle viability was indirectly assessed by testing whether muscles we re able to contract in response to a specific electrical stimulation protocol (1 train/sec, train duration of 250 ms, 50 Hz, 10 ms delay, 10 ms duration, 30 V). Muscles were then electrically stimulated at 10 Hz, 50 V, 10 ms pulses with 90 ms delays for 10 min. while the contralateral ED L from each animal served as a non-contracting control. Buffer was refreshed 3 times at the e nd of the contraction period, and muscles were incubated with the cell impermeable fluorescent dete ctor of nitric oxide, 4,5-diaminofluorescein (DAF-2, 0.02 M, Cayman Chemical Company, Ann Ha rbor, MI) in the dark for 10 min. Aliquots of buffer were analyzed for DAF fluorescence using a spectrofluorometer with excitation wavelength at 485 nm and emissi on wavelength at 538 nm. Absorbances were normalized to EDL wet weight. To test for DAF-2 specificity, a separate experiment was conducted using both EDL and soleus muscles from the same animal. In this experiment both EDL musc les were electrically stimulated, while the soleus muscles were not. The protocol consisted of an initial 10 min incubation, after which the inhib itor of NOS, L-NMMA (1 mM), was added to the buffer of one EDL and one soleus. Muscles were incubated with L-NMMA for 10 min, followed by 10 min of contraction (EDL muscles) or re sting (soleus muscles). Buffer wa s refreshed 3 times afterwards,

PAGE 36

36 followed by a 10 min incubation with DAF-2. Aliqouts of buffer were analyzed spectrophotometrically for DAF fluorescence. We measured an ~10% increase in background fluorescence in the soleus muscles induced by L-NMMA, and a tota l prevention of the contraction-induced fluorescence in the EDL mu scles with L-NMMA. Thus, DAF-2 specificity was confirmed in this model. Fiber Isolation The gastrocnemius complex, consisting of the plantaris, soleus, and medial and lateral heads of the gastrocnemius, was carefully re moved from both hindlimbs. The plantaris and soleus were removed, weighed, and frozen in liquid nitrogen for further analyses. The remaining gastrocnemius muscle was rinsed in PBS, cu t down the middle, weighed, and incubated in DMEM containing 0.02% collagenase, type I (Wor thington, Lakewood, NJ). Both gastrocnemius muscles per animal were incubated in a 15-ml Falcon tube for 1.5 h at 37oC with gentle shaking. After this incubation, the digest was carefully triturated three tim es, and incubated an additional 10 min. The collagenase was then diluted by allowi ng the digest to settle for approximately 5 min. Most of the media was removed and fresh media without collagenase was added. This was repeated for a total of 3 times. The resulting digested muscles were decanted into p-100s containing DMEM. Intact, is olated fibers were selected using a pipet. Only fibers that were free of other cellular debris were se lected and plated. These viable fibers were plated in 24-well plates with growth factor-re duced matrigel (BD Biosciences Bedford, MA) and serum free media. Occasionally, emanating cel ls of non-muscle origin (nonspindle-shaped) were evident and fibers containing these cells were excluded from analyses. For the HGF experiment, fibers were cultu red with 0.002% BrdU and HGF in serum free media for 48 h, and fixed for sa tellite cell emanation quantific ation, and BrdU immunostaining. For the ADMA experiments, fibers were incubate d with ADMA at concentrations of 0.1-50 M

PAGE 37

37 for 24 h in serum free media. Then, 0.002% BrdU wa s added and the plates were centrifuged at 1500 x g for 30 min to induce satell ite cell activation. The fibers we re cultured for an additional 24 h and then fixed for quantification and BrdU immunostaining. For the final fiber experiment, L-arginine and DETA-NO were added to the media with 0.002% BrdU immediately before centrifuging at 1500 x g for 30 min to induce satellite cell activati on. Fibers were cultured for 48 h and fixed for cell emanation quantification and BrdU immunostaining. A subset of fibers with emanating cells was stained for myoD to confirm that emanating cells were of muscle origin. Fibers stained for pax7 were plated in 24-well plates and fixed immediately. Immunohistochemistry To measure satellite cell activation, the numbe r of cells staining positive for BrdU (BrdU+) were counted. Fixed fibers were in cubated in 1 M HCl for 45 min at 45oC. This was neutralized with 0.1 M borate buffer (pH 8.5) for 10 min. Fibers were blocked in 5% goat serum for 1 h, followed by incubation in primary anti-BrdU an tibody (1:75, Megabase Research, Lincoln, NE) for 2 hours. They were then incubated in secondary anti-rabbit-Texas Red antibody (1:100, Vector Laboratories) for 1 hour and staine d with DAPI-containing mounting media (Vector Laboratories). Fibers st ained for pax7 were permeabilized with 0.1% Triton-X for 5 min. and blocked in 5% goat serum for 1 h. Primary anti-pax7 (R&D Systems, Minneapolis, MN) was applied for 1 h at 1:50 dilution. Secondary anti -mouse-Texas Red (1:100) was incubated for 1 h. Fibers were mounted with DAPI-containing mounti ng media. All fibers were viewed on a Zeiss microscope with rhodamine, FITC, and DAPI filters. To confirm that the spindle-shaped emana ting cells (i.e. mononuclear cells growing on the substratum adjacent to myofibers 48 h after cent rifugation) were of musc le origin, a subset of cultures was stained for myoD using the Tyra mide Signal Amplification (TSA) kit (Perkin Elmer, Boston, MA). Fixed cultures were incubated in 3% H2O2 to quench endogenous

PAGE 38

38 peroxidases. Fibers were bl ocked in TNB blocking buffer ( 0.1 M Tris-HCl, 0.15 M NaCl, 0.5% Blocking Reagent, pH 7.5) for 1 h, followed by incubation in prim ary anti-myoD antibody (1:200, Novo Castra, UK) for 2 h. They were then incubated in secondary anti-mousebiotinylated antibody (1:400, Pierce ) for 1 h. Cultures were incubate d with streptavidin-HRP for 30 min, followed by fluorophore tyramide working solution for 5 min. Finally, cultures were mounted with DAPI-containing mounting media (Vect or Laboratories). Greater than 95% of these emanating cells were myoD+, confirming that the spindleshaped cells adjacent to the myofibers were emanating cells from those fibers. Asymmetric Dimethylarginine (ADMA) ELISA ADMA was measured in serum and muscle (plantaris) homogenate samples using an ELISA kit (Alpco Diagnostics, Salem, NH) accordi ng to the kit instructions. The kit is a more convenient method to measure ADMA than ex isting techniques, such as HPLC, liquid chromatography-mass spectrometry, and gas chro matography-mass spectrometry, but correlates well with these other methods (49). Western Blotting Plantaris muscle samples were homogenized in 50 mM Tris (p H 7.5), 150 mM NaCl, 5 mM EDTA, 50 mM sodium phosphate, 15% glycerol 1mM benzamidine, 10 mg/ml pepstatin A, 5 g/ml leupeptin, 1 mM PMSF, 2 M DTT, and 1 g/ml aprotinin. Homogenates were centrifuged for 10 min at 1,000 g to remove connective tissue a nd cellular debris and protein content was quantified in the supe rnatant using the DC Protein A ssay Kit (Bio-Rad Laboratories, Richmond, CA). Forty micrograms of total pr otein were fractionate d by SDS-PAGE and electroblotted onto polyvinylid ene difluoride (PVDF) membranes. Membranes were then blocked for 1 h in Tris-buffe red saline -Tween 20 (TBS-T) (TrisHCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20) containing 5% nonfat dry milk at room temperature, and then incubated with

PAGE 39

39 primary antibody (nNOS 1:500, e NOS 1:500) overnight at 4oC. Membranes were washed three times in TBS-T, 10 min each, followed by incubation with peroxidase-labeled anti-rabbit or antimouse antibodies (Vector Laborator ies, Burlingame, CA) for 1 h at room temperature. Reactions were developed by using the enhanced chemilu minescence detection reagents (ECL Plus; Amersham Biosciences, Buckinghamshire, UK), according to the manufacturer's instructions, and protein levels were determined by densitometr y (Scion Image software). Blots were stripped (Restore Stripping Buffer, Pier ce Chemical) and reprobed for -actin (1:8000, Abcam) to control for loading errors. Statistical Analysis Data were analyzed by a two-way ANOVA (age x treatment) with Tukeys HSD post hoc test. Data from Experiment 4 (ADMA treatment of young fibers only), western blots, and pax7 data were analyzed by a one-way ANOVA. Si gnificance was established a priori at p<0.05. Values reported are means SEM.

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40 CHAPTER 4 RESULTS Muscle Atrophy in Male C57 Mice We found evidence of both maturation and senescence effects when comparing muscle masses and the ratio of muscle mass to body ma ss between our three age groups (Table 4-1). Specifically, muscle masses for the gastrocnemius, plantaris, soleus, and ti bialis anterior (TA) significantly increased between ages 2 and 10 months, representing maturation effects. The muscle mass to body mass ratio for the gastrocnemiu s, plantaris, and TA significantly decreased between these ages. Senescence effects were noted as a significant decrease in gastrocnemius, plantaris, and soleus masses between ages 10 and 22 months. In addition, the muscle mass to body mass ratio of the soleus sign ificantly decreased between ag es 10 and 22 months. The total protein per plantaris muscle was significantly in creased from 2 month to 10 months, while total protein per gram of wet weight was not different between ages. Nitric Oxide Production from Contracting Muscle Since nitric oxide (NO) is a primary signa l for satellite cell activation (2, 4, 50, 52), we sought to determine whether NO production was re duced from aged muscle and whether this could account for the previously reported deficits in satellite cell activ ity (53, 61) and muscle repair (17, 19, 20, 43). NO production, as qua ntified by DAF fluorescence immediately following a 10-min contraction protocol, was significantly lower from adult and old EDL muscles as compared to young muscles (Figure 4-1). NO production did not differ between adult and old EDL muscles. This finding was unexpected since deficits in satellite cell activity are not evident in the adult muscles, compared to young. A ll ages exhibited a significant increase in NO production following contraction, and the fold cha nge over resting was not significantly reduced

PAGE 41

41 in aged and adult EDL muscles compared to young, suggesting that NO production is not limiting in adult or aged muscles. Nitric Oxide Synthase Protei n Expression Is Not Reduced Next, we sought to determine whether protei n expression of nitric oxide synthases (NOS) was decreased in aged muscle. Both constituti vely active NOS isoforms, nNOS and eNOS, were expressed equally in plantaris homogenate from young, adult, and aged muscle (p = 0.907 for nNOS and p=0.976 for eNOS, Figures 4-2 and 43, respectively). Thus, a reduction in NOS protein content cannot explai n the decreased NO production fr om adult and aged muscle. Asymmetric Dimethylarginine Can Inhibit Satellite Cell Activity We found that inhibition of NOS activity with the competitive inhibitor, asymmetric dimethylarginine (ADMA), prevented satellite ce ll activation, as quantif ied by both satellite cell emanation (Figure 4-4A) and BrdU incorporatio n (Figure 4-4B), from young mouse myofibers (Figure 4-5A-D). In all isolated myofibers expe riments, satellite cell activation was assessed by quantifying emanating cells, and positive immunostai ning for BrdU. In a subset of fibers with emanating cells, greater than 95% of these cells were myoD+, confirming that the spindle-shaped cells adjacent to the myofib ers were emanating from those fibers. ADMA doses of 0.1-50 M reduced satellite cell activation of young myofibers exposed to th e inhibitor for 24 h prior to mechanical stimulation. Both 10 and 50 M ADMA significantly reduced satellite cell emanation from young fibers (Figure 4-5A), while 50 M ADMA significantly reduced BrdU incorporation compared to control (Figure 4-5C). However, even the lowest dose (0.1 M) negatively affected satellite cel l activation on young fibers. Our fi ndings suggest that inhibition of NOS activity with the competitive inhibitor, ADMA, is sufficient to attenuate satellite cell activation from young fibers expos ed to mechanical stress.

PAGE 42

42 Asymmetric Dimethylarginine Is Not El evated in Aged Muscle and Blood Next, we measured the levels of endogenous asymmetric dimethylarginine (ADMA) in the serum and plantaris homogenate of young and old animals. ADMA levels in the muscle and serum did not differ between young and old mi ce (Table 4-2), thereby eliminating an accumulation of ADMA as a potential cause of decreased NO production with age. Aged Myofibers Have Equal Numbers of Pax7+ Satellite Cells We quantified the number of pax7+ nuclei/fiber by immunostaining myofibers immediately fixed after isolation and plating (F igure 4-6). There were no significant differences between the mean number of pax7+ nuclei per fiber for any age group (Young = 3.16 0.296, Adult = 3.08 0.384, Old = 2.79 0.321). This is important because it confirms that any deficits in satellite cell activation are not due to fewer satellite cells present initially on aged fibers. Satellite Cell Response to HGF Is Not Altered with Age To determine whether the deficit in aged satellite cell activation is upstream of HGF release, we examined the response of satellite cells on isolated myofibers to various doses of HGF. Adult and aged satellite cells reacted simila rly to young satellite cell s in response to HGF (Figure 4-7A-D). Regardless of age, 10-20 ng/ml of HGF significantly increased satellite cell activation (as quantified by satellite cell emana tion and BrdU incorporat ion 48 h post-plating). Fifty ng/ml HGF did not significan tly increase BrdU incorporati on above control levels for any age. This finding implies that there is an uppe r limit to HGFs effects on satellite cell activation. However, satellite cells from adult and aged fibers respond to HGF in the same manner as young satellite cells, suggesting that the aging deficit lies upstream of HGF release. L-Arginine and a Nitric Oxide Donor Ca n Rescue Aged Satellite Cell Activity The similar response of satelli te cells to an HGF dose response suggests the aging deficit lies upstream of HGF rel ease. Since NO triggers HGF release, we sought to determine whether

PAGE 43

43 the NOS substrate, L-arginine, or a NO donor (DETA-NO) could res cue satellite cell activation from mechanically stimulated aged myof ibers. Myofibers were treated with L-arginine (2 mM) or DETA-NO (10 M) and centrifuged to stimulate activ ation of satellite cells. Adult satellite cell activation (satelli te cell emanation and BrdU inco rporation 48 h post-plating) mirrored young satellite cell activity with both treatments. Satellite cell activation from aged myofibers was improved with L-arginine a nd DETA-NO (Table 4-3 and Figure 4-8 A-D). However, both treatments increased satellite ce ll activity in young and a dult fibers as well. Importantly, young, adult, and old control fibers, in the absence of mechan ical stress, were not significantly different when comparing satellite cell emanation or BrdU incorporation. There was a trend for centrifugation to stimulate sate llite cell emanation in young (p=0.074) and adult (p=0.065) fibers, while this was not true for old fibers (p=0.788). To confirm these results were due to NO produc tion and not indirect effects of the parent compound, we tested a dose response of NO-depleted DETA (5-50 M) and found no increase in satellite cell activation above control levels (data not show n). In sum, both the NOS substrate and the NO donor were able to rescue satellite cell activity from aged muscle to above young control levels, but were not able to normalize aged satellite cell activity to levels measured in young myofibers with the same treatment.

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44 Table 4-1. Body mass, gastrocnemius mass, musc le mass/body mass ratios, and total protein for young, adult, and old mice. Values represent mean SEM. Total protein measurements were made for plantaris muscle.*Significantly different from Young. #Significantly different from Adult. Young (2 mo) Adult (10 mo) Old (22 mo) Body Mass (g) 24.77 0.58 34.63 1.38* 29.17 0.95*# Gastrocnemius Mass (mg) 126.92 2.45 160.40 2.14* 131.05 2.887# Mass/Body Mass 5.13 0.069 4.67 0.117* 4.50 0.063* Plantaris Mass (mg) 17.88 0.49 21.58 0.50* 18.21 0.46# Mass/Body Mass 0.72 0.016 0.63 0.026* 0.63 0.013* Protein (mg/g w.w.) 4.58 0.143 5.06 0.236 5.00 0.349 Total Protein (mg) 0.86 0.046 1.07 0.055* 0.93 0.088 Soleus Mass (mg) 10.02 0.32 13.55 0.45* 9.61 0.34# Mass/Body Mass 0.40 0.011 0.40 0.018 0.33 0.014*# Tibialis anterior Mass (mg) 49.98 1.64 59.28 0.86* 55.68 1.61* Mass/Body Mass 2.02 0.060 1.73 0.057* 1.92 0.067

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45 Table 4-2. Asymmetric dimethylar ginine (ADMA) concetration ( mol/L) in serum and muscle of young and old mice. Values represent mean SEM. Muscle ADMA was measured in plantaris homogenate. Young (2 mo) Old (22 mo) Serum 1.46 0.14 0.82 0.15 Muscle 6.18 0.87 5.00 0.44

PAGE 46

46 Table 4-3. Quantification of satellite cell activa tion as measured by (A) cell emanation and (B) BrdU incorporation from young, adult, and old myofibers 48 h post-plating. Values represent mean SEM for n = ~50-90 fibe rs/group. Centr.=Centrifuged for 30 min at 1500 g *Significantly different from Y oung Control and Young Centrifuged. #Significantly different from Young Centrifuged + Arginine. Significantly different from Young Centrifuged + DETA-NO. Significantly different from Age-Matched Centrifuged. Significantly different from Adult Control. Significantly different from Adult Centrifuged. Significantly different from A dult Centrifuged + Arginine. Significantly different from A dult Centrifuged + DETA-NO. Significantly different from Old Control. A. Young (2 mo) Adult (10 mo) Old (22 mo) Control 0.18 0.059 0.16 0.056 0.19 0.088 Centrifuged 0.42 0.068 0.42 0.069#, 0.24 0.072#,,, L-Arg + Centr. 1.04 0.101*, 1.18 0.115*,, 0.74 0.103*,#,,, ,,,, DETA-NO + Centr. 1.30 0.101*,#, 1.38 0.111*,#,, 0.73 0.091*,#,,, ,,,, B. Young (2 mo) Adult (10 mo) Old (22 mo) Control 0.21 0.08 0.26 0.11 0.22 0.09 Centrifuged 0.28 0.061 0.27 0.065 0.19 0.058#,,, L-Arg + Centr. 0.60 0.079 0.56 0.084 0.44 0.087 DETA-NO + Centr. 0.79 0.085*, 0.73 0.089*,, 0.60 0.083

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47 Figure 4-1. Nitric oxide (NO) production as quantified by 4, 5-diaminofluorescein (DAF) fluorescence following 10 min of in vitro EDL muscle contraction. Values are normalized to EDL muscle wet weight. Valu es represent mean SEM. *Significantly different from Young Resting, p<0.05. #Significantly different from Age-Matched Resting, p<0.05. +Significantly different from Young Contracting, p<0.05.

PAGE 48

48 A. nNOS Young Adult Old Beta-actin Young Adult Old B. Figure 4-2. Protein expression of neuronal nitric oxide synthase (nNOS). (A) Representative immunoblot of nNOS from young, adult, and ol d plantaris muscle. Beta-actin served as a loading control. (B) Quantification of immunoblots for nNOS normalized to beta-actin. Values represent mean SEM.

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49 A. eNOS Young Adult Old Beta-actin Young Adult Old B. Figure 4-3. Protein expression of endothelial nitric oxide syntha se (eNOS). (A) Representative immunoblot of eNOS from young, adult, and ol d plantaris muscle. Beta-actin served as a loading control. (B) Qu antification of immunoblots for eNOS normalized to betaactin. Values represent mean SEM.

PAGE 50

50 A. B. Figure 4-4. Representative images of activated sate llite cells. (A) Representa tive light image of a satellite cell emanating from an isolated mouse myofiber indicated by white arrow (120x magnification). (B). Representative fluo rescent image of a satellite cell staining positive for BrdU (red). Nuclei are staine d blue with DAPI (60x magnification).

PAGE 51

51 B. C. D. Figure 4-5. Response of fibers to asymmetric di methylarginine (ADMA). (A) Mean satellite cell emanation from young fibers and (B) percentage of young fibers with emanating satellite cells following exposure to varyi ng concentrations of ADMA 24 h prior to mechanical centrifugation ( 1500g, 30 min). (C) Mean BrdU+ cells per fiber and (D) percentage of fibers with BrdU+ nuclei following exposure to varying concentrations of ADMA 24 h prior to mechanical centrif ugation. Values represent mean SEM of ~50 fibers/group. *Significantly different from 0 M ADMA.

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52 Figure 4-6. Representative image of a fiber stained for pax7 (red) immediately following isolation and plating (~30 fibers per age group). White arrows indicate pax7+ nuclei. Blue = DAPI-stained nuclei, Red = pax7+ nuclei (60x magnification).

PAGE 53

53 A. B. C. D. Figure 4-7. Response of fibers to hepatocyte gr owth factor (HGF). (A) Mean satellite cell emanation per fiber and (B) percentage of fibers with emanating satellite cells in response to varying concentra tions of HGF. (C) Mean BrdU+ cells per fiber and (D) percentage of fibers with BrdU+ nuclei following exposure to varying concentrations of HGF. Approximately 50 fi bers/group were analyzed.

PAGE 54

54 A. B. Figure 4-8. Response of fibers to L-arginine and DETA-NO. (A) Mean satellite cell emanation per fiber and (B) percentage of fibers with emanating satellite cells in response to treatment with 2 mM L-arginine or 10 M DETA-NO and centrifugation (1500g, 30 min). (C) Mean BrdU+ cells per fiber and (D) percentage of fibers with BrdU+ nuclei following exposure to L-arginine and D ETA-NO and centrifugation. Approximately 90 fibers per group were analyzed. (A.) and (C.) represent mean SEM. *Significantly different from Young Control. +Significantly different from AgeMatched Control. #Significantly different from Young Centrifuged. Significantly different from Age-Matched Centrifuged.

PAGE 55

55 Figure 4-8 Continued. C. D.

PAGE 56

56 CHAPTER 5 DISCUSSION Main Findings Although our old mice were in the early stages of senescence, based on survival curves for this strain, we found evidence of muscle atrophy in the gastrocn emius, plantaris, and soleus muscles. Single myofibers were isolated from the gastrocnemius muscles with resident satellite cells remaining. We studied the activation of these satellite cells, de fined as myogenic cell emanation from the fiber and BrdU incorporation at 48 h post-plating. The main findings of this study are: 1.) Satellite cell activa tion in response to mechanical s timulation is reduced with age; 2.) Nitric oxide (NO) and L-arginine suppl ementation effectively increase satellite cell activation; however, an age-related deficit remains; 3.) In vitro NO production is reduced from adult and aged contracting mu scle; and 4.) Aged fibers respond normally to exogenous hepatocyte growth factor (HGF). The data indi cate that the aging deficit in satellite cell activation lies in the sensitivity of aged satellite cells to NO. Reduced Satellite Cell Activation in Re sponse to Mechanical Perturbation In response to a mechanical stress (centrifugati on), the increase in dividing cells emanating from aged fibers was reduced compared to young, and fewer nuclei stained positive for BrdU (Table 4-3 and Figure 4-8 control vs. centrifuged, across supplement groups). These data depict the deficit in satellite cell activation from old mu scle. Nevertheless, there were no differences in pax7+ satellite cells present on young, adult, and old fibers fixed immediatel y after isolating, in agreement with several human (29, 47) and animal studies (42). In contrast, many animal studies indicate that the sate llite cell population declin es with aging (5, 12, 28, 51). Differences in muscles studied, ages of animals, and methods for identifying satellite cells may explain the differences in findings. Unlike most aging stud ies, our old group (22 mo) was younger than the

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57 50% mortality age for this strain (27 mo). Ther efore, it is possible that the decline in the quiescent satellite cell population reported by other auth ors occurs only during advanced senescence. Our animals, which were in earl y senescence, exhibit impaired satellite cell responsiveness to extern al stimuli, without a decline in the overall quies cent population. Our data imply that there is a deficit in th e signaling pathways lead ing to activation of resident satellite cells on old muscle. This pathway involves a sequence of events including: 1) mechanical perturbation, 2) nitric oxide release, 3) HGF release fr om the extracellular matrix, 4) HGF binding to the c-met receptor on satellite ce lls, and finally, 5) activ ation of DNA synthesis and migration of satellite cells (Figure 5-1). We studied the mechanism of age-associated satellite cell dysfunction by examining satellite cell activation in isolated myofibers following experimentally induced mechani cal perturbation (centrifugation) exogenous NO treatment, or exogenous HGF application, in order to localize the aging deficit. L-Arginine and a Nitric Oxide Donor Ca n Rescue Aged Satellite Cell Activity The NOS substrate, L-arginine, or an NO donor, DETA-NO, res cued satellite cell activation in mechanically stimulated aged myof ibers, in that satellite cell activation from old fibers exposed to these treatments exceeded the le vel of satellite cell act ivation in young control fibers (Table 4-3 and Figure 4-8). We confir med that the DETA-NO e ffects were due to NO production and not indirect effects of the pa rent compound, DETA, by ex posing fibers to NOdepleted DETA (5-50 M). These data suggest that an age-related reduction in NO production could contribute to reduced sa tellite cell activity. However, both L-arginine and DETA-NO treatments also increased satelli te cell activity in young and adult fi bers such that satellite cell activity in old fibers remained significantly lo wer than in young and adult fibers with the Larginine or DETA-NO treatments. It appears that NOS activity/ NO production may limit satellite

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58 cell activation from all ages since exogenous NO s upplementation increased satellite cell activity across age groups. Our centrifugation protocol was a submaximal s timulus that did not induce 100% satellite cell activation. Consequently, the degree of satellite cell activation following a fixed stimulus (i.e., centrifugation or exposur e to a fixed dose of DETA-NO or L-arginine) indicates the sensitivity of the system to that particular stimulus. Therefore, in spite of the potential therapeutic effects of augmenting NO production, we must conc lude that age-related deficits responsible for compromised satellite cell activation lie downstream of this event. Other groups have completely re stored satellite cell activation/ proliferation in old muscle with exposure to young serum (20), forced Notch activation (19), IGF-1 (17), or basic FGF2 (51). However, these researchers studied both ac tivation and proliferation. Our experimental design, on the other hand, focused primarily on the act ivation step, since fibe rs were fixed before significant proliferation occurred. We conclude that the aging deficit in satellite cell activation lies in the sensitivity of aged satellite cells to NO. Nitric Oxide Production and NOS Prot ein Expression from Aged Muscle Nitric oxide is a primary signal for HGF release leading to satellite ce ll activation (2, 4, 50, 52). Therefore, we hypothesized that NOS expression and/or NO production w ould be reduced in aging skeletal muscle. Myofiber isolation re quired both gastrocnemius muscles, leaving insufficient tissue for additional measurements. Therefore, in vitro contraction-induced NO production was measured in the EDL, and NOS protein expression in the plantaris. These muscles were chosen since they resemble the gast rocnemius in fiber type (primarily type II) and location (hindlimb). NO production, as quant ified by DAF fluorescence following muscle contraction, was significantly lo wer in aged and adult, compared to young EDL muscle (Figure 4-1). While all ages exhibited an increas e in NO production following contraction, the young

PAGE 59

59 EDL muscles produced significantly more NO followi ng contraction than adult or aged muscles. However, the fold-change over resting values wa s not significantly reduc ed in adult or aged muscles, suggesting that contraction-induced NO production is not limiting in aged animals. Ours is the first study providing evidence of a functional decrease in NO production from aged, as well as adult muscle. The reduced NO production from adult muscle was unexpected since no reduction in satellite cell responses were noted in adult fibers compared to young (Figure 4-8). We found no differences in n NOS and eNOS protein expression between young, adult, and old plantaris muscles (Figures 42, 4-3). Thus, down-regulation of NOS enzyme expression may not explain the reduced NO producti on from adult and old EDL muscles. We did not measure NOS activity, but Richmonds and colleagues (46) found a reduction in nNOS activity and percentage of NOS-c ontaining fibers in muscles from aged rats (24 mon) compared to young (8 mon). In contrast, Capanni and coll eagues (15) reported an increase in nNOS expression in 24 month-old rat gracilis muscle However, this group measured NOS protein expression via immunoblots and immunohistoche mistry, whereas Richmonds and colleagues (46) measured functional NOS activity. It is pos sible that NOS enzymatic activity is inhibited even with an upregulation of nN OS protein expression (15), or no change in NOS expression as we show. While it is an interesting observation that NO production is decreased in adult and aged muscle compared to young, it does not appear that a significant loss of contraction-induced NO production by the EDL is associated with sene scence (i.e., no difference between adult and old muscles). Therefore, reduced NO pr oduction is unlikely to explain agerelated deficits in satellite cell activation in these muscles.

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60 Satellite Cell Response to HGF Is Not Altered with Age We determined that the deficit in aged sate llite cell activation lies upstream of HGF release since the response of aged sate llite cells on isolated myofiber s to a dose response of HGF was similar to young satellite cells. Regardless of age, 10-20 ng/ml of HGF significantly increased satellite cell activation (satellite cell emanation and BrdU incorporation 48 h post-plating), in agreement with previously published studies (5 5, 58). Fifty ng/ml HGF did not significantly increase BrdU incorporation above control levels for any age. This finding implies that there is an upper limit to HGFs effect s on satellite cell activation. The similar response of young, adult, and old sate llite cells is important because it implies that all events downstream of HGF release, such as HGF/c -met colocalization and c-met signaling, are normal in aged muscle and satellite cells. In contrast, Barani and colleagues (5) reported a lower increase in the HGF receptor, c-met, expression in cultured muscle-derived cells (mdc) of old rats with serum stimulation co mpared to young. Young (3 week-old) and adult (9 month-old) mdc cultures increase d relative c-met expression by ~190% with serum stimulation. In contrast, old (24 month-old) mdc increase d c-met expression by just 130% with serum stimulation. However, the cell surface density of the c-met receptor was increased to the same extent in the three ages with serum exposure. Therefore, similar responses to a fixed dose of HGF, as we observed, are not surprising. These obse rvations effectively lo calize the aging deficit in satellite cell activation to the mechanisms mediating HGF release in response to NO production. Asymmetric Dimethylarginine Is Not Elevated in Aged Muscle and Blood but Does Inhibit Satellite Cell Activity To explain the aging deficit in satellite cell activation a nd the reduction in NO production with age, we studied the levels of ADMA, a competitive inhibitor of NOS, in the serum and

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61 plantaris homogenate of old a nd young mice, and its effects on satellite cell activity. Conboy and colleagues (20) used a series of in vivo and in vitro experiments to show that factors in old serum negatively influence satellite cell activation, proliferation, and musc le repair. However, components in young serum alone are sufficient to re store satellite cell function in aged muscle. Their data imply that the old syst emic environment is altered such that there is a decrease in a positive factor found in young serum, an increase in a negative or inhibitory factor, or both. We hypothesized that ADMA was that negative factor found at high levels in old serum. As a NOS inhibitor, it may potentially i nhibit satellite cell activity. We found that ADMA doses of 0.1-50 M negatively impacted satellite cell activation (Figure 4-5). Myofibers from young muscles were adversely affected by ADMA such that 10 and 50 M significantly reduced satellite ce ll emanation from fibers, while 50 M ADMA significantly reduced BrdU incor poration compared to control. Our findings provide further evidence of the importance of NO for satellite cell activation since ADMA is sufficient to attenuate satellite cell activity. However, we did not measure an increase in ADMA in old animals. ADMA levels were similar in the serum and muscle homogenate of young and old animals (Table 4-2), suggesting that ADMA concentration and subsequent NOS inhibition is not elev ated with age. One group of researchers has reported an incr ease in ADMA in serum from aged (20 mo) rats compared to young (6 mo) animals (60). Seru m L-arginine levels were similar between the two ages, so the L-arginine/ADMA ratio was lowe r in old rats compared to young. Lastly, serum concentrations of nitrite were significantly decreased in these old animals. While ADMA is an appealing candidate for the reduction in NO produc tion with age, we we re unable to provide evidence of an accumulation of AD MA in old mice. Nevertheless, our data showing that ADMA

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62 inhibits satellite cell activity on fibers confirm th e integral role of NO signaling for satellite cell activation from quiescence. Response to Nitric Oxide Under conditions where satelli te cells are kept quiescent, muscle produces NO in low level pulses (56, 58). On the other hand, mechan ical stress, associated with satellite cell activation, produces a bolus of NO via nNOS acting as a mechanotransducer (56). NO is important for triggering the release of HGF from the extracellular matrix (ECM) to activate satellite cells (2, 4, 50, 52). Tatsumi et al. (54, 55) have demonstrated th at release of HGF, in response to mechanical perturba tion, is NO-dependent. When rele ased, HGF rapidly co-localizes with its receptor on satellite cells, the c-met tyrosine kinase recepto r, and triggers activation from quiescence (53). Once activated, sa tellite cells synthesize both HGF and c-met (3, 53). HGF is synthesized as a precursor prot ein requiring proteolytic cleavag e to produce the active growth factor. However, Tatsumi and Allen (52) confirme d that active heterodimeric HGF is present in the ECM of uninjured rat muscle, indicating that proteolytic cleavage to activate HGF is not a rate-limiting step in satellite cel l activation, but rather the steps leading to release of active HGF from its ECM tethering. Yamada and colleagues (62) recently provi ded evidence of the involvement of matrix metalloproteinases (MMPs) in th e NO-dependent release of HGF in vitro Inhibition of MMPs along with treatments that induce satellite cell activation (stretc h, NO supplementation) inhibited activation of cultured ra t satellite cells. In addition, conditioned medi um from satellite cells exposed to activating stimuli plus the inhibitor of MMPs did not activate cultured sate llite cells and did not contain HGF. Importantly, the MMP inhibitor did not inhibit NOS activity. These data suggest that MMPs cleave HGF from the ECM a nd that this step is tr iggered by NO release.

PAGE 63

63 Barani et al. (5) measured MMP 2-9 activ ity on crushed muscle extract (CME), and found that these activities were reduced with aging. MMP2-9 enzyme activity measured in initial muscle derived cells (mdc) was identical in young (3 week-old) and old (24 month-old) rats, and serum exposure increased activity uniformly in both ages. However, when measured on CME, MMP 2-9 enzyme activity was ~2-fold lower in old CME compared to young. This may explain the aging deficit in satellite cell activation and warrants furthe r investigation. Another possible explanation for the aging defic it in satellite cell activation is a decr ease in endogenous HGF levels with aging. If HGF production goes down with age, there would be less present to stimulate activation in response to NO signaling. Thus, we postulate that an age-related decline in MMP activity and/or a reduction in HGF expressi on decreases the sensitivity of satellite cells to NO, thus limiting satellite cell activation. Limitations and Future Directions The age of the old mice (22 mo) may have li mited the severity of the aging deficit in satellite cell activation in response to mechanical perturba tion. According to the National Institute on Aging (NIA), the ag e of 50% mortality for male C 57 mice is 27 months. Using mice 27 months or older may have amplified the effects of our treatments on satellite cell activity. On the other hand, the presence of a deficit in satellite cell activat ion during early senescence (i.e., 22 mo) highlights the potential c linical signifi cance of this phenomenon in an aging population. Higher doses of arginine and DETA-NO may have improved satellite cell activation from old fibers. These doses were selected based on dose responses in young muscle. It may be that higher doses were necessary to maximally stimul ate satellite cell activa tion on old fibers. Plus, our mechanical stimulus (centrifugation, 1500 g 30 min) was a submaximal stimulus for activation. Using a more severe mechanical pertur bation and/or higher dos es of supplements may have activated more satellite cells and eliminat ed aging deficits. Nevertheless, the submaximal

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64 stimuli chosen allows assessment of the sensitivit y of the satellite cell activation system. Future studies should focus on physiologically-relevant ac tivating stimuli, such as stretch or injury. Isolated myofibers are extremely useful fo r studying satellite cells in their resident position on the myofiber. However, it is difficult to harvest fibers for such assays as Western blotting or PCR. Thus, we were limited to im munostaining of fibers to identify and count satellite cells. Future studies should include trea tment of the whole animal with L-arginine or NO donors to allow for more robust measurements. We did not measure NOS activity in the gastro cnemius to confirm that it is reduced with aging. NOS protein expression in the plantaris was not changed with aging, but NO production was decreased in contracting adult and aged EDL muscle. We cannot determine whether NO production by isolated gastrocnemius myofiber s during centrifugation was reduced. Regardless, reduced NO production does not seem to be the cause of the aging deficit in satellite cell activation since this deficit persists with administration of a fi xed amount of NO donor. Lastly, we did not find a reduction in pax7+ cells per fiber between our ages. However, due to the limitation of available fibers, onl y ~30 fibers from each age were stained and analyzed. Although there did not appear to be a trend for reduced satell ite cells in the aging gastrocnemius, the variability between fibers wi thin an age group, and even within one mouse, was high. Staining more fibers, such as 100 fibe rs per age group, may have shown a significant loss of satellite cells with age. Future studies need to confirm equal pax7+ nuclei per fiber with this strain of mouse and these ages. Conclusions We have successfully narrowed the aging defi cit in satellite cell ac tivation in response to mechanical perturbation to the steps betw een NO production and HGF release. Based on our data, NO production does not limit satellite cell ac tivity in old muscle, an d the steps downstream

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65 of HGF are not impaired in old fibers. Figure 5-1 diagrams the signaling events leading to satellite cell activation from quiescence. We conclude that the deficit in activation lies between NO production and HGF release, in the sens itivity of old sate llite cells to NO.

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66 Figure 5-1. Overview of the steps leading to sate llite cell activation and th e potential location of the aging deficit. Satellite Cell Activation (DNA Synthesis and Migration) HGF/c-met co-localization and Signaling HGF Release from ECM Mechanical Perturbation NO Release (Location of Aging Deficit)

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67 LIST OF REFERENCES 1. Alway SE, Love DA, and Chen KD The effects of age and hindlimb suspension on the levels of expression of the myogenic regulator y factors MyoD and m yogenin in rat fast and slow skeletal muscle. Exp Physiol 86: 509-517, 2001. 2. Anderson JE A role for nitric oxide in muscle repa ir: NO-mediated satel lite cell activation. Mol Biol Cell 11: 1859-1874, 2000. 3. Anderson JE Studies of the dynamics of skeletal muscle regeneration: the mouse came back! Biochem Cell Biol 76: 13-26, 1998. 4. Anderson JE and Pilipowicz O Activation of muscle satellite cells in single fiber cultures. Nitric Oxide 7: 36-41, 2002. 5. Barani AE; Durieux A, Sabido O, and Freyssenet D Age-related changes in the mitotic and metabolic characteristics of muscle-derived cells. J Appl Physiol 95: 2089-2098, 2003. 6. Baumgartner RN, Koehler KM, Gallagher D, Romero L, Heymsfield SB, Ross RR, Garry PJ, and Lindeman RD Epidemiology of sarcopenia among the elderly in New Mexico. Am J Epidemiol 147: 755-763, 1998. 7. Bischoff R Proliferation of muscle satellite cells on intact myofibers in culture. Dev Biol 115: 129-139, 1986a. 8. Bischoff R A satellite cell mitogen from crushed adult muscle. Dev Biol 115: 140-147, 1986b. 9. Bischoff R Analysis of muscle regeneration using single myof ibers in culture. Med Sci Sports Exerc 21: S164-S172, 1990a. 10. Bischoff R Cell cycle commitment of rat muscle satellite cells. J Cell Biol 111: 201-207, 1990b. 11. Bischoff R. Chemotaxis of skeletal muscle satellite cells. Dev Dyn 208: 505-515, 1997. 12. Bockhold KJ, Rosenblatt JD, and Partridge TA Aging normal and dystrophic mouse muscle: analysis of myogenicity in cultures of living single fibers. Muscle Nerve 21: 173183, 1998. 13. Booth FW. Regrowth of atrophied skeletal muscle in adult rats after ending immobilization. J Appl Physiol 44: 225-230, 1978. 14. Brack AS, Bildsoe H, and Hughes SM. Evidence that satellite cell decrement contributes to preferential decline in nuclear number from la rge fibres during murine age-related muscle atrophy. J Cell Sci 118: 4813-4821, 2005.

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68 15. Capanni C, Squarzoni S, Petrini S, Villanova M, Muscari C, Maraldi NM, Guarnieri C, and Caldarera CM. Increase of neuronal nitric oxide synthase in rat skeletal muscle during aging. Biochem Biophys Res Commun 245(1): 216-219, 1998. 16. Chakravarthy MV, Booth FW, and Spangenburg EE. The molecular responses of skeletal muscle satellite cells to con tinuous expression of IGF-1: implications for the rescue of induced muscular atrophy in aged rats. Int J Sport Nutr Exerc Metab 11: S44-S48, 2001. 17. Chakravarthy MV, Davis BS, and Booth FW. IGF-1 restores satellite cell proliferative potential in immobilized old skeletal muscle. J Appl Physiol 89: 1365-1379, 2000. 18. Childs TE, Spangenburg EE, Vyas DR, and Booth FW. Temporal alterations in protein signaling cascades during rec overy from muscle atrophy. Am J Physiol Cell Physiol 285: C391-C398, 2003. 19. Conboy IM, Conboy MJ, Smythe GM, and Rando TA. Notch-mediated restoration of regenerative potential to aged muscle. Science 302: 1575-1577, 2003. 20. Conboy IM, Conboy MJ, Wagers AJ, Gir ma ER, Weissman IL, and Rando TA Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature 433: 760-764, 2005. 21. Conboy IM, and Rando TA. The regulation of notch signa ling controls satellite cell activation and cell fate determin ation in postnatal myogenesis. Dev Cell 3: 397-409, 2002. 22. Cooke JP. Asymmetrical dimethylarginine. The uber marker? Circulation 109: 1813-1819, 2004. 23. Crameri RM, Langberg H, Magnusson P, Jensen CH, Schroder HD, Olesen JL, Suetta C, Teisner B, and Kjaer M. Changes in satellite cells in human skeletal muscle after a single bout of high in tensity exercise. J Physiol 558: 333-340, 2004. 24. Darr KC, and Schultz E. Exercise-induced satellite cell ac tivation in growing and mature skeletal muscle. J Appl Physiol 63(5): 1816-1821, 1987. 25. Desplanches D. Structural and functional adaptations of skeletal muscle to weightlessness. Int J Sports Med 18(Suppl 4): S259-S264, 1997. 26. Edstrom E, and Ulfhake B. Sarcopenia is not due to lack of regenerative drive in senescent skeletal muscle. Aging Cell 4: 65-77, 2005. 27. Gallegly JC, Turesky NA, Strotman BA, Gurley CM, Peterson CA, and DupontVersteegden EE. Satellite cell regulation of muscle mass is altered at old age. J Appl Physiol 97: 1082-1080, 2004.

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69 28. Gibson MC and Schultz E. Age-related differences in absolu te numbers of skeletal muscle satellite cells. Muscle Nerve 6: 574-580, 1983. 29. Hikida RS, Walsh S, Barylski N, Camp os G, Hagerman FC, and Staron RS. Is hypertrophy limited in elderly muscle fi bers? A comparison of elderly and young strength-trained men. Basic Appl Myol 8: 419-427, 1998. 30. Johnson SE and Allen RE. Proliferating cell nuclear antig en (PCNA) is expressed in activated rat skeletal muscle satellite cells. J Cell Physiol 154(1): 39-43, 1993. 31. Kadi F, Charifi N, Denis C, and Lexell J. Satellite cells and m yonuclei in young and elderly women and men. Muscle Nerve 29: 120-127, 2004. 32. Kim JS, Cross FM, and Bamman MM. Impact of resistance loading on myostatin expression and cell cycle regulati on in young and older men and women. Am J Physiol Endocrinol Metab 288(6): E1110-1119, 2005. 33. Leikert JF, Rathel TR, Muller C, Vollmar AM, and Dirsch VM Reliable in vitro measurement of nitric oxide re leased from endothelial cells using low concentrations of the fluorescent probe 4, 5-diaminofluorescein. FEBS Lett. 506(2):131-134, 2001. 34. Lexell J Human aging, muscle mass, and fiber type composition. J Gerontol A Biol Sci Med Sci 50: 11-16, 1995. 35. Machida S and Booth FW Regrowth of skeletal musc le atrophied from inactivity. Med Sci Sports Exerc 36: 5259, 2004. 36. Marsh DR, Criswell DS, Carson JA, and Booth FW Myogenic regulatory factors during regeneration of skeletal muscle in young, adult, and old rats. J Appl Physiol 83: 12701275, 1997. 37. Mauro A. Satellite cells of skeletal muscle fibers. J Biophys Biochem Cytol 9: 493-495, 1961. 38. Mitchell PO and Pavlath GK. A muscle precursor cell-depende nt pathway contributes to muscle growth after atrophy. Am J Physiol Cell Physiol 281: C1700-C1715, 2001. 39. Mitchell PO and Pavlath GK. Skeletal muscle atrophy leads to loss and dysfunction of muscle precursor cells. Am J Physiol Cell Physiol 287: C1753-C1762, 2004. 40. Naka D, Yoshiyama IT, Miyazawa K, Hara H, Hishida T, and Kitamura N. Activation of hepatocyte growth factor by proteolyti c conversion of single chain form to a heterodimer. J Biol Chem 267: 20114-20119, 1992. 41. Nakamura T, Teramoto H, and Ichihara A. Purification and characterization of a growth

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70 factor from rat platelets for mature parenchymal hepatocytes in primary cultures. Proc Natl Acad Sci USA 83: 6489-6493, 1986. 42. Nnodim JO. Satellite cell numbers in senile rat levator ani muscle. Mech Ageing Dev 112: 99-111, 2000. 43. Pattison JS, Folk LC, Madsen RW, and Booth FW. Identification of differentially expressed genes between young and old rat soleus muscle during recovery from immobilization-induced atrophy. J Appl Physiol 95: 2171-2179, 2003. 44. Rathel TR, Leikert JF, Vollmar AM, and Dirsch VM. Application of 4,5diaminofluorescein to reliabl y measure nitric oxide released from endothelial cells in vitro Biol Proced Online 5: 136-142, 2003. 45. Renault V, Thornell LE, Eriksson PO, Butler-Browne GS, and MoulyV. Regenerative potential of human skelet al muscle during aging. Aging Cell 1: 132-139, 2002. 46. Richmonds CR, Boonyapisit K, Kusner LL, and Kaminski HJ. Nitric oxide synthase in aging rat skeletal muscle. Mech Ageing Dev 109: 177-189, 1999. 47. Roth SM, Martel GF, Ivey FM, Lemmer JT Metter EJ, Hurley BF, and Rogers MA. Skeletal muscle satellite cell populations in healthy young and older men and women. Anat Rec 260: 351-358, 2000. 48. Sajko S, Kubinova L, Cvetko E, Kreft M, Wernig A, and Erzen I. Frequency of Mcadherin-stained satellite cells decl ines in human muscles during aging. J Histochem Cytochem 52: 179-185, 2004. 49. Schulze F, Wesemann R, Schwedhelm E, Sy dow K, Albsmeier J, Cooke JP, and Boger RH. Determination of asymmetric dimethyl arginine (ADMA) using a novel ELISA assay. Clin Chem Lab Med 42: 1377-1383, 2004. 50. Sheehan SM, Tatsumi R, Temm-Grove CJ, and Allen RE HGF is an autocrine growth factor for skeletal muscle satellite cells in vitro. Muscle Nerve 23: 239-245, 2000. 51. Shefer G, Van de Mark DP, Richar dson JB, and Yablonka-Reuveni Z. Satellite-cell pool size does matter: defining the myogenic potency of aging skeletal muscle. Dev Biol 294: 50-66, 2006. 52. Tatsumi R and Allen RE. Active hepatocyte growth fact or is present in skeletal muscle extracellular matrix. Muscle Nerve 30: 654-658, 2004. 53. Tatsumi R, Anderson JE, Nevoret CJ, Halevy O, and Allen RE. HGF/SF is present in normal adult skeletal muscle and is capable of activating satellite cells. Dev Biol 194: 114-128, 1998.

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71 54. Tatsumi R, Hattori A, Ikeuchi Y, Anderson JE, and Allen RE. Release of hepatocyte growth factor from mechanically stretched skeletal muscle sa tellite cells and role of pH and nitric oxide. Mol Biol Cell 13(8): 2909-2918, 2002. 55. Tatsumi R, Sheehan SM, Iwasaki H, Hattori A, and Allen RE. Mechanical stretch induces activation of skeletal muscle satellite cells in vitro. Exp Cell Res 267: 107-114, 2001. 56. Tidball JG, Lavergne E, Lau KS, Spencer MJ, Stull JT, and Wehling M. Mechanical loading regulates NOS expressi on and activity in developing and adult skeletal muscle. Am J Physiol 275: C260-C266, 1998. 57. Wagers AJ, and Conboy IM. Cellular and molecular signature s of muscle regeneration: current concepts and controversies in adult myogenesis. Cell 122: 659-667, 2005. 58. Wozniak AC, and Anderson JE. Nitric oxide-dependence of sa tellite stem cell activation and quiescence on normal skeletal muscle fibers. Dev Dynamics 236: 240-250, 2007. 59. Wozniak A, Pilipowicz O, Yablonka-Reuveni Z, Greenway S, Craven S, Scott E, and Anderson JE. C-met expression and mechanical activ ation of satellite cells on cultured muscle fibers. J Histochem Cytochem 51: 1-9, 2003. 60. Xiong Y, Yuan LW, Deng HW, Li YJ, and Chen BM. Elevated serum endogenous inhibitor of nitric oxide synthase an d endothelial dysfunction in aged rats. Clin Exp Pharm Physiol 28: 842-847, 2001. 61. Yablonka-Reuveni Z, Seger R, and Rivera AJ. Fibroblast growth factor promotes recruitment of skeletal muscle sa tellite cells in young and old rats. J Histochem Cytochem 47: 23-42, 1999. 62. Yamada M, Tatsumi R, Kikuiri T, Okamoto A, Nonoshita S, Mizunoya W, Ikeuchi Y, Shimokawa H, Sunagawa K, and Allen RE. Matrix metalloprotei nases are involved in mechanical stretch-induced activation of skeletal muscle satellite cells. Muscle Nerve 34: 313-319, 2006.

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72 BIOGRAPHICAL SKETCH Jenna Betters was born in New Smyrna Beach, Florida, in 1979. She was salutatorian of her high school graduating class in 1997. She received her bachel ors degree in biology in 2001 from the University of North Florida in Jacks onville, Florida, where she was a member of Phi Kappa Phi Honor Society, University Scholars Honor Society, and Golden Key International Honor Society. Jenna obtained her Master of Sc ience in Exercise and Sport Sciences from the University of Florida in 2004. Her thesis was ti tled Trolox supplementation during mechanical ventilation attenuates contractil e dysfunction and protein degrada tion. She received a Graduate Alumni Fellowship, allowing her to pursue the Do ctor of Philosophy degree. She has worked as a research assistant in the Mo lecular Physiology Laboratory with in UFs Center for Exercise Science since 2001. After graduating, Jenna plans to continue working in the area of skeletal muscle research.


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