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This is to certify that the

thesis entitled

Satellite Cell Proliferation and Differentiation
During Postnatal Growth of Porcine Skeletal Muscle

presented by

Nicholas T. Mesires

has been accepted towards fulfillment
of the requirements for

Food Science &
Human Nutrition

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SATELLITE CELL PROLIFERATION AND DIFFERENTIATION DURING
POSTNATAL GROWTH OF PORCINE SKELETAL MUSCLE

BY

Nicholas T Mesires

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

2000

ABSTRACT

SATELLITE CELL PROLIFERATION AND DIFFERENTIATION DURING
POSTNATAL GROWTH OF PORCINE SKELETAL MUSCLE

By
Nicholas T Mesires

DNA accretion is essential to skeletal muscle growth and is a result of
satellite cell proliferation, differentiation and fusion with existing muscle fibers.
The objective of this study was to quantify populations of proliferating and
differentiating satellite cells throughout porcine skeletal muscle growth. Satellite
cells were isolated from the semitendinosus (ST) muscle of pigs at 1, 7, 14, and
21 weeks of age, separated from cellular debris and immunostained. Satellite
cells were quantified by positive staining for a muscle-specific neural cell
adhesion molecule (NCAM). Antibodies against proliferating cell nuclear antigen
(PCNA) and myogenin were employed to quantify proliferating and differentiating
satellite cells, respectively. Increases in ST muscle weight were closely
paralleled by increases in total DNA and protein. The proportion of satellite cells
to non-myogenic cells as well as the rate of differentiation decreased from 1 to 7
weeks. A modest decline in the percentage of proliferating satellite cells
occurred from 1 to 21 weeks with a large proportion (>75%) remaining
proliferative at 21 weeks. These results indicate that a high percentage of
satellite cells remain proliferative throughout postnatal growth and the rate of

differentiation may be a critical regulatory point for skeletal muscle growth.

This thesis is dedicated to my parents who have always believed in me and have

given me every opportunity to be the best I could be. This is also for Kristin who

is my inspiration and has been there with love and encouragement every step of
the way.

ACKNOWLEDGMENTS

I am indebted to a number of people who have in one way or another
contributed to this project. First of all, I would like to thank my major professor,
Dr. Matt Doumit for his guidance, friendship as well as challenging me both in the
laboratory and the classroom. He has also gone above and beyond the call of
duty for an advisor by being a active member of the lab and helping to collect
even the most tedious of data (e.g. cell counting). I would also like to thank the
members of my guidance committee (Dr. Dale Romsos, Dr. Gale Strasburg and
Dr. George Smith) for their valuable input and advice both for this thesis as well
as for my graduate program. I would also be remiss if I did not acknowledge the
efforts of Dr. Rob Templeman in providing assistance with the statistical analysis
of the data.

I owe special thanks to Ms. Sharon Debar for her assistance with lab
work. She has always made herself available to me and has been an immense
help, especially with those famous “last-minute experiments”. I appreciate all of
the time and effort she has put into these projects.

I would like to also thank Torn Forton, Jennifer Dominguez and the Meat
Lab crew for slaughtering and disposal of the pigs used in this study as well as
being patient while we took the samples we needed. I would also like to thank Al
Snedegar and the MSU Swine Farm for providing the right animals at the right
time.

I would like to thank my lab mates, Chuck Allison, John Heller and Scott

Kramer for a lot of good times as well as some thoughtful criticism along the way.

I would also like to thank the undergraduates who have worked in the lab and
whose willingness to help out with this project has not gone unnoticed. They are
Betsy Booren, Matt Ritter, Sally Dean, and Jeannine Grobbel. I appreciate all of
the work you guys have done!

Lastly, I would like to thank Kristin who has been my toughest critic at
times and has done it with love and encouragement. I would also like to thank

her for here willingness to help with the statistical analysis and SAS programs.

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. vi
LIST OF FIGURES ................................................................................ vii
LIST OF ABBREVIATIONS ..................................................................... ix
INTRODUCTION .................................................................................... 1
CHAPTER ONE
Literature Review ................................................................................. 3
l. Satellite Cells and Skeletal Muscle Growth ............................................. 3
ll. Age-related Changes in Satellite Cell Activity .......................................... 9
III. Effects of Growth Factors on Satellite Cell Activity ................................... 15
A. Hepatocyte Growth Factor (HGF) .................................................... 15
B. Fibroblast Growth Factor-2 (FGF-2) ................................................. 18
C. Insulin-Like Growth Factor-l (IGF-I) .................................................. 22
CHAPTER TWO
Quantification of Proliferating and Differentiating Satellite
Cells in Growing Pigs From 1 to 21 Weeks of Age ................................... 25
I. ABSTRACT .................................................................................... 25
II. INTRODUCTION .............................................................................. 26
III. MATERIALS AND METHODS ............................................................ 28
A. Animals ..................................................................................... 28
B. Satellite Cell Isolation ................................................................... 28
C. Tissue Collection ......................................................................... 30
D. DNA Assay ................................................................................. 31
E. Biuret Protein Assay ..................................................................... 32
F. Culture of 5.1 H11 and F50 Hybridoma Cell Lines ............................... 32
G. Satellite Cell Enrichment and Adsorption to Coverslips ........................ 34
H. lmmunostaining for Neural Cell Adhesion Molecule (NCAM) ................. 35
I. Immunostaining for Proliferating Cell Nuclear Antigen (PCNA) .............. 36

vi

J. Immunostaining for Myogenin ......................................................... 37

K. Statistical Analysis ....................................................................... 37
IV. RESULTS

A. Live Weight and Muscle Weights ...................................................... 38

B. DNA and Protein Contents .............................................................. 38

C. Percentage of Satellite Cells (NCAM+ cells) ....................................... 39

D. Percentage of Proliferating Satellite Cells (NCAM+/PCNA+ cells) ............ 39

E. Percentage of Proliferating Non-myogenic Cells

(NCAM-IPCNA+ cells) ................................................................... 40

F. Percentage of Differentiating Satellite Cells (myogenin+ cells) ............... 40
V. DISCUSSION .................................................................................. 41
VI. CHAPTER TWO TABLES AND FIGURES ............................................. 49
CHAPTER THREE
Recommendations for Future Research ................................................ 78
REFERENCES .................................................................................... 81

vii

LIST OF TABLES

CHAPTER TWO

Table 2.1. Antibodies used to quantify proliferation and
differentiation of porcine satellite cells ....................................................... 51

viii

LIST OF FIGURES

' CHAPTER ONE

Figure 1.1. Regulatory Checkpoints in Satellite Cell Activity ........................... 7

CHAPTER TWO

Figure 2.1. Liveweight (kg) of growing pigs at 1, 7, 14,
and 21 weeks of age ............................................................................. 53

Figure 2.2. Left semitendinosus muscle weight (9), total DNA
(mg) and total protein (9) of growing pigs at 1, 7, 14, and
21 weeks of age ................................................................................... 55

Figure 2.3. Right Iongissimus dorsi muscle weight (9), total
DNA (mg) and total protein (9) of growing pigs at 1, 7, 14,
and 21 weeks of age .............................................................................. 57

Figure 2.4. Daily myonuclear accretion of the left
semitendinosus muscle of growing pigs from 1 to 7, 7 to 14
and 14 to 21 weeks of age ...................................................................... 59

Figure 2.5. Protein to DNA ratio of the left semitendinosus
and right Iongissimus dorsi muscles of growing pigs at 1, 7,
14 and 21 weeks of age ......................................................................... 61

Figure 2.6. Fluorescent micrograph of porcine satellite cells
stained for the presence of neural cell adhesion molecule (NCAM) .................. 63

Figure 2.7. Fluorescent micrograph of porcine satellite cells
stained for the presence of proliferating cell nuclear antigen (PCNA) ............... 65

Figure 2.8. Fluorescent micrograph of porcine satellite cells
stained for the presence of myogenin ....................................................... 67

Figure 2.9. Fluorescent micrographs depicting representative
non-specific staining of porcine satellite cells to NCAM, PCNA
and myogenin ...................................................................................... 69

ix

Figure 2.10. Percentage of satellite cells (cells expressing NCAM)
isolated from the right semitendinosus muscle of growing pigs at
1, 7, 14, and 21 weeks of age .................................................................. 71

Figure 2.11. Percentage of proliferating satellite cells (cells

expressing both PCNA and NCAM) isolated from the right

semitendinosus muscle of growing pigs at 1, 7, 14, and 21

weeks of age ....................................................................................... 73

Figure 2.12. Percentage of non-myogenic proliferating cells

(cells expressing PCNA but not NCAM) isolated from the right

semitendinosus muscle of growing pigs at 1, 7, 14, and 21

weeks of age ....................................................................................... 75

Figure 2.13. Percentage of differentiating satellite cells (cells
expressing myogenin) isolated from the right semitendinosus
muscle of growing pigs at 1, 7, 14, and 21 weeks of age ............................... 77

LIST OF ABBREVIATIONS

3H-Thymidine: Tritiated thymidine

ACTH: Adrenocorticotropic hormone

BrdU: 5-bromo-2’-deoxyuridine

CK: Creatine kinase

CME: Crushed muscle extract

CNR: Cytoplasmic volume to nucleus ratio
DAPI: 4’,6-diamidino—2-phylindole

DNA: deoxyribonucleic acid

EGF: Epidermal growth factor

ELISA: Enzyme-linked immunosorbent assay
FBS: Fetal bovine serum

FGF-2: Fibroblast growth factor-2 or basic fibroblast growth factor
FITC: Fluorescein isothiocyanate

HGF: Hepatocyte growth factor

HM: Heavy muscled

IGFBP: Insulin-like growth factor binding protein

xi

lGF-l: Insulin-like growth factor I

IGFR-1: Insulin-like growth factor receptor-1
LD: Iongissimus dorsi
LM: Light muscled
MAPK: Mitogen-activated protein kinase
M-CSF: Macrophage colony-stimulating factor
MEM: Minimum essential medium
MHC: Myosin heavy chain
mRNA: Messenger ribonucleic acid
NCAM: Neural cell adhesion molecule
PBS: Phosphate buffered saline
PCNA: Proliferating cell nuclear antigen
PDGF: Platelet-derived growth factor
RNA: Ribonucleic acid
SEM: Standard error of the mean
ST: semitendinosus

TRITC: Tetramethyl rhodamine isothiocyanate

xii

INTRODUCTION

Elucidation of the cellular mechanisms governing skeletal muscle growth
will be beneficial for development of strategies to increase the efficiency of lean
tissue growth in food animal species. Increases in efficiency of animal growth
would also lessen the burden of these production systems on financial and
environmental resources. Furthermore, such discoveries may provide insight
into the treatment of human diseases such as muscular dystrophy.

Skeletal muscle growth occurs through hyperplasia (an increase in muscle
fiber number) and hypertrophy (an increase in muscle fiber size). Since the
majority of hyperplasic growth occurs before birth (Stickland et al., 1975;
ngmore and Stickland, 1982), optimization of postnatal skeletal muscle growth
must occur through manipulation of mechanisms regulating muscle hypertrophy.
Skeletal muscle hypertrophy is accompanied by DNA accretion in individual
fibers and increases in the protein synthesis capacity of the muscle (Powell and
Aberle, 1975; Cheek et al., 1970, 1971). Satellite cells, first discovered by
Alexander Mauro in 1961, are the sole source of DNA that is added during
postnatal skeletal muscle hypertrophy (Schultz and McCormick, 1994). Satellite
cells develop as a discrete myogenic cell type in the later stages of. embryonic
development (Stockdale, 1992), but do not terminally differentiate and fuse with
developing muscle fibers as do embryonic myoblasts. Since muscle fiber nuclei
are incapable of further DNA synthesis (Stockdale and Holtzer, 1961), satellite
cells function to provide DNA for the postnatal growth and regeneration of muscle

fibers (Campion, 1984). Satellite cells proliferate to produce a population of

daughter cells, a subset of which differentiate and fuse with existing muscle
fibers to facilitate muscle hypertrophy. The remaining undifferentiated cells
continue to proliferate or become quiescent to be available for future growth or
regeneration (Moss and LeBlond, 1971).

Satellite cells exhibit a high degree of proliferation at birth, but the
percentage of nuclei in skeletal muscle that synthesize DNA declines in the later
stages of life as these cells become mitotically quiescent (Schultz and Lipton,
1982; Mulvaney et al., 1988; Mozdziak et al., 1994). Although satellite cells have
been shown to differentiate and fuse (Stockdale and Holtzer, 1961; Moss and
LeBlond, 1971), it is unknown if the in vivo rate of differentiation changes
throughout postnatal growth. Characterization of these indices of satellite cell
activity will provide insight into the mechanisms of DNA accretion and muscle
hypertrophy as well as the age at which application of growth promoting agents
to growing animals may be the most beneficial.

A variety of hormones and growth factors influence satellite cell activity
and muscle hypertrophy (Allen and Rankin, 1990; Dodson et al., 1996), yet the
interactions of these growth factors and the mechanisms by which they affect
myogenesis in vivo are unknown. Furthermore, the levels of these growth factors
present in muscle throughout postnatal life and their effects on growth are
unknown. Therefore, identification and characterization of the pertinent growth
factors regulating skeletal muscle growth may lead to advances in the

development of new strategies to optimize skeletal muscle growth.

CHAPTER 1

LITERATURE REVIEW

Satellite Cells and Skeletal Muscle Growth

Since muscle fiber number is determined prenatally and remains constant
throughout life, postnatal growth occurs by hypertrophy of existing muscle fibers
(Stickland et al., 1975; Wrgmore and Stickland, 1982, 1983). A number of
studies have shown that postnatal muscle hypertrophy is dependent upon DNA
accretion. Powell and Aberle (1975) established this association through
comparison of heavy muscled (HM) and light muscled (LM) growing pigs. Heavy
muscled pigs exhibited a greater biceps femoris muscle mass, as well as a
greater total DNA content than LM pigs. These data indicate that greater rates of
hypertrophy of muscle are dependent upon accretion of DNA. These findings are
corroborated by studies of Cheek et al. (1970, 1971), who concluded that a
muscle nucleus can accommodate a fixed cytoplasmic volume. In other words,
there is a fixed ratio of DNA to protein. Taken together, these studies
demonstrate that DNA accretion is necessary to increase the protein synthetic
capacity of muscle leading to fiber hypertrophy and ultimately muscle growth.

It is generally accepted that nuclei situated under the sarcolemma of
muscle fibers, termed myonuclei, lose the ability to synthesize DNA. In a study
by Stockdale and Holtzer (1961), multinucleated myotubes were examined both
in vitro and in vivo to determine the source of incorporated nuclei. These authors

observed that a 30-minute exposure of embryonic chick muscle to tritiated

thymidine (3H-Thymidine) labeled only mononucleated cells that were situated
outside of the sarcolemma, indicating that only these nuclei were synthesizing
DNA. Identical treatment of myotube cultures in vitro failed to label any nuclei
that had been incorporated into a myotube. However, mixed myoblast/myotube
cultures pulse labeled for 30 minutes, washed and kept in culture for an
additional 4 days did produce labeled nuclei within myotubes, demonstrating
fusion of mononucleated cells that had been synthesizing DNA at the time of 3H-
Thymidine exposure into myotubes. This study demonstrated that myotube
formation occurs through fusion of multiple mononucleated cells, not through
mitotic or amitotic division of nuclei already located in the myotubes. In a similar
experiment, nuclei incorporated into a myotube failed to incorporate 3’H-
Thymidine yet myotubes were still able to accumulate DNA to facilitate a
regeneration response (Snow, 1978). These studies demonstrate that DNA
accretion of skeletal muscle fibers occurs through incorporation of
mononucleated cells that reside outside of the sarcolemma.

At birth, mononucleated cells continue to reside beneath the basement
membrane of individual muscle fibers yet remain outside of the sarcolemma
(Stockdale, 1992; Mauro, 1961). These mononucleated cells were termed
satellite cells and were first discovered in the tibialis anticus muscle of the frog by
Alexander Mauro in 1961. Satellite cells consist of a heterochromatic nucleus,
scant cytoplasm and few organelles (Mauro, 1961; White and Esser, 1989). The
developmental time point in which satellite cells and embryonic myoblasts

diverge and become separate myogenic cell lines is unknown (Campion, 1984).

One hypothesis postulates that satellite cells are remnants of embryonic
myoblasts that do not terminally differentiate and are carried forward and
encased within the basement membrane. Another plausible hypothesis
speculates that satellite cells and embryonic myoblasts diverge as separate
myogenic lineages when pluoropotent stem cells undergo determination (White
and Esser, 1989; Schultz and McCormick, 1994).

Satellite cells increase the DNA content of existing muscle fibers through
proliferation, followed by differentiation and fusion of these daughter cells with
the fibers. In a seminal study by Moss and LeBlond (1971), young Sherman rats
(14-17 days of age) received a single intraperitoneal injection of 3H-Thymidine,
and were sacrificed 1 to 72 hours after injection. Transverse sections of tibialis
anterior muscle were analyzed to quantify those nuclei that incorporated the label
as well as the position of those nuclei. Satellite cells were found to be the only
cell type to incorporate the label at the earliest time points, the percentage of
which increased to 10 hours post-injection. An increase in labeled myonuclei
was observed at 24 and 48 hours, while the percentage of labeled satellite cells
decreased at these time points. Results of Allbrook et al. (1971), corroborate the
above findings through quantification of the degree to which satellite cells
incorporate 3H-Thymidine. One hour after injection, satellite cells exhibited a
high degree of labeling (4-8 silver grains per nucleus). At 24 hours post-injection
the average number of silver grains per satellite cell nucleus is reduced (1-4
silver grains per nucleus), with a concommitant increase in the number of labeled

myonuclei. These data provide conclusive evidence that postnatal DNA

accretion occurs through proliferation of satellite cells, followed by the
differentiation and fusion of daughter cells with existing muscle fibers.
Furthermore, daughter cells that do not terminally differentiate provide for a
“reserve” population for future cycles of proliferation and differentiation.

Satellite cells are usually mitotically quiescent in mature uninjured muscle
(White and Esser, 1989), yet can be recruited into the cell cycle following a wide
range of events including injury, denervation, exercise, overuse (Schultz and
McCormick, 1994) or during growth (Moss and Leblond, 1970). Satellite cell
activity is regulated in part at the transition from quiescence to proliferation
(Figure 1.1.). For example, treatment of quiescent satellite cells in vitro with
hepatocyte growth factor (HGF) causes satellite cells to have a shortened lag
phase of growth when compared to control cultures. This indicates a more rapid
transition of cells from Go to G1 phase of the cell cycle (Allen et al., 1995). When
quiescent satellite cells from the female Ievator ani muscle are exposed to
testosterone, proliferation is induced as measured by uptake of 3H-Thymidine
(Joubert and Tobin, 1995). Yablonka-Reuveni et al. (1999), demonstrated that
treatment of muscle fiber associated satellite cells with fibroblast growth factor-2
(FGF-2) induced satellite cells to enter the cell cycle as measured by expression
of proliferating cell nuclear antigen (PCNA). Since satellite cells will remain
quiescent when cultured in association with the muscle fiber (Bischoff, 19863),
these data demonstrate that satellite cell activity is in part regulated at the

transition from quiescence.

 

 

Quiescent Satellite Cell

Regulation Point #1:
Transition from Quiescence to
Proliferation

Regulation Point #2: 5.3:
Rate of Proliferation

 

    
 

Regulation Point #3:
Rate of Differentiation

 

Differentiating
Satellite Cell

 

Myonuclei
Muscle

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Figure 1.1.TRegulatory Checkpoints in Satellite Cell Activity

The rate of proliferation is another checkpoint at which satellite cell activity
is regulated (Figure 1.1.). The cell division cycle is made up of a 1) a pre-
synthetic gap phase (G1 phase), 2) a DNA synthesis phase (S phase), 3) a post-
synthetic gap phase (G; phase), and 4) a mitosis phase (M phase). While S, M,
and G2 are relatively constant, the G1 phase appears to be the most variable.
Hence, the rate by which a satellite cell proliferates depends upon the time it
takes to progress through G1 (Allen, 1979). Turkey satellite cell clones isolated
from the same animal have been shown to have different kinetics of proliferation
under identical culture conditions (McFarland et al., 1995). Cell cycle time for
satellite cells in 30—day-old growing rats has been estimated to be approximately
32 hours in length with an S phase of 14 hours (Schultz, 1996). In these rats,
approximately 80% of satellite cells divide within a 32-hour cell cycle time as
measured by 5-bromo-2’-deoxyuridine (BrdU) incorporation. The remaining 20%
divide at a much slower rate, possibly entering a quiescent state before
undergoing another division. Taken together, these data indicate that the rate of
proliferation is a mechanism by which satellite cell activity may be modulated.

The rate at which satellite cells differentiate is another regulatory
checkpoint for satellite cell activity (Figure 1.1.). Changes in the rate of
differentiation affect the subsequent fusion of satellite cells with existing muscle
fibers as well as the number of satellite cells that remain proliferating. Many
growth factors and hormones may exert their effects in part through a decrease
in differentiation (reviewed by Allen and Rankin, 1990; Dodson et al., 1996).

Doumit et al. (1996) demonstrated that in vitro treatment of porcine satellite cell

clones with 10'7M testosterone caused a decrease in differentiation as measured
by a decreased percentage (relative to total nuclei) of myotube nuclei, nuclei
expressing myosin heavy chain (MHC) protein, as well as a decrease in creatine
kinase (CK) activity. These researchers postulated that a decline in satellite cell
differentiation would result in an expanded population of proliferative satellite

cells.

Age-related Changes in Satellite Cell Activity

Age-related changes in total satellite cell numbers and proliferative
capacity have been reported previously. The percentage of total nuclei that were
classified as satellite cells in the subclavius and pemneus muscles decreases
from 30-35% in the neonate to about 5% in mature rats (Allbrook et al., 1971).
Using colchicine to induce metaphase arrest, the incidence of mitotic figures in
10-week-old rats was roughly half of that in 3-week-old rats, indicating a reduced
proliferative capacity as a function of age. These data are corroborated by
studies of Cardasis and Cooper (1975), who reported a similar decline in the
number of satellite cell nuclei in the gastrocnemis muscle of growing mice.

The peroneus Iongus and sarton'us muscles of the pig also exhibit a
decrease in the percentage of satellite cells as a function of age. The
percentage of total nuclei classified as satellite cells decreased from
approximately 15% in 1-week-old pigs to less than 5% in 64-week-old pigs
(Campion et al., 1981). As in other studies, the percentage of total nuclei

classified as myonuclei increased to 64 weeks of age, indicating DNA accretion

and hypertrophy of the muscles studied. However, these data suggest that
absolute number of satellite cells may have increased from 1 to 32 weeks of age
and was at least maintained from 1 to 64 weeks of age. Campion et al. (1982)
reported that total numbers of satellite cells also increased from 4 days to 4
weeks of age in the semimembranosus muscle of both fast growing and normal
Japanese quail. An increase in the total numbers of satellite cells has been
observed in the soleus muscle of 1 to 12-month-old rats but not in the extensor
digitorum longus muscle at similar ages (Gibson and Schultz, 1983).
Collectively, it appears that a postnatal increase in absolute numbers of satellite
cells is possible even if the proportion of satellite cells to total myonuclei declines
throughout growth. Furthermore, the changes in the total number of satellite
cells throughout growth depend in part on the muscles examined.

In vitro studies have also demonstrated age-related changes in satellite
cell proliferation. When rat satellite cells are isolated and plated at clonal
densities, the average number of cells per colony is inversely proportional to the
age of the animal from which the cells were isolated (Schultz and Lipton, 1982).
These data indicate that the proliferative capacity of satellite cells in vitro is
dictated in part by the age of the animal. Similar differences are observed when
satellite cells are plated in mass culture. Primary rat satellite cells isolated from
3-week-old rats have a shorter lag phase of PCNA expression in vitro when
compared to cells isolated from 9-month-old rats (Johnson and Allen, 1993).
Yablonka-Reuveni et al. (1999) demonstrated delayed expression of PCNA in

muscle fiber-associated satellite cells isolated from 10-month-old rats when

10

compared to that of 3-week-old rats. These studies provide indirect evidence for
an age-related decline in satellite cell proliferation.

Age-related differences have also been reported using muscle
damage/regeneration models to study satellite cell activity. Six and 16-week-old
rats were subjected to treadmill exercise to induce focal damage in the soleus
muscle. 5-bromo-2’-deoxyuridine labeling index in sections of the m. soleus
reached a maximum at 24 hours and 168 hours post-exercise in 6-week and 16-
week-old rats, respectively (Jacobs et al., 1995). When rat primary satellite cell
cultures are induced to proliferate by exposure to low-level irradiation, 3H-
Thymidine incorporation decreased with the age of rat from which the cells where
isolated (Ben-Dov et al., 1999). These studies demonstrate a reduced capacity
of cells undergoing proliferation to facilitate a regeneration response due to age.

The age-related changes in satellite cell activity are also influenced by the
physiological status of the animal. Gonadally intact male pigs, castrated males,
and castrated males implanted with testosterone propionate were sacrificed at 7,
14, and 21 days of age after a 6-hour continuous infusion of 3H-Thymidine
(Mulvaney et al., 1988). Myofiber segments were isolated from the triceps
brachii muscle and total and labeled nuclei were enumerated. An age-related
decline in satellite cell DNA synthesis was observed under each physiological
condition. The rate of decline was greatest for castrated pigs, whereas the
decline was attenuated in gonadally intact pigs and pigs treated with testosterone
propionate. Clonally derived turkey satellite cells exhibit an age-related decline in

proliferation when exposed to serum from hens at 3 to 15 weeks of age, but not

11

to serum from tom turkeys (Doumit et al., 1990). These data indicate that an
age-related decrease in satellite cell activity may occur relatively early in
postnatal life and may be modulated by hormonal status of the animal.
Post-hatch turkey skeletal muscles also exhibit age-related changes in
satellite cell activity (Mozdziak et al., 1994). Tom turkeys at 3, 6, 9, 18, and 26
weeks of age were infused with BrdU to measure DNA synthesis. Myofiber
segments with associated satellite cells were enzymatically isolated from the
pectoralis thoracicus and biceps femoris muscles and stained for the presence of
BrdU. Myofiber diameter and cytoplasmic volume to nucleus ratio (CNR) were
also measured. Myofiber diameter increased throughout the entire experimental
period, indicating muscle hypertrophy. From 3 to 6 weeks of age, CNR
increased in association with a high degree of DNA synthesis, expressed as the
number of BrdU labeled nuclei per 1000 total nuclei. Between 6 and 9 weeks of
age, no changes occurred in CNR, but DNA synthesis declined to low levels. At
later stages of growth (9-26 weeks of age), CNR increased steadily whereas
DNA synthesis continued to decline (Mozdziak et al., 1994). These data indicate
that DNA synthesis is important relatively early in turkey skeletal muscle growth
and that increases in CNR constitute the majority of the hypertrophy response in
the later stages of growth. When satellite cell DNA synthesis is abolished through
irradiation in the left pectoralis thoracicus muscle of 3-week-old turkeys, muscle
weight is depressed up to 15 weeks after treatment when compared to contra-
Iateral control (Mozdziak et al., 1997). Irradiation also caused a depression in

the number of nuclei per myofiber length due to a decrease in DNA synthesis at

12

1 and 4 weeks after irradiation. Since CNR is not different at any time after
treatment, these data demonstrate that inhibition of high levels of DNA synthesis
at 3-6 weeks of age depresses subsequent myonuclear accretion and muscle
growth.

Relatively few data exist on the effects of age on satellite cell
differentiation and fusion. A study by Allen et al. (1982) examined the effect of
donor age on the ability of satellite cell-derived myotubes to accumulate muscle-
specific proteins in vitro. Satellite cells were isolated and cultured from neonatal
rats (<5 days of age), growing rats (1-3 months of age), adult rats (9-12 months
of age) and old rats (>24 months of age), allowed to differentiate and incorporate
into myotubes in vitro, then assayed for accumulation of a-actin protein. Cultures
from neonatal rats were shown to accumulate significantly more a-actin than
growing, adult or old rats, but no significant differences were observed between
these later ages. Although this study did not measure differentiation directly, it
appears that muscle-specific protein accumulation does not vary during the later
stages of postnatal growth.

In vitro measures also have been used to determine if age-related
changes in satellite cell differentiation exist. Satellite cells were isolated from the
pectoralis major muscle of 3, 9, and 15-week-old turkeys, grown to near-
confluence in culture, trypsinized and passaged. Tertiary culture satellite cells
derived from 9 and 15-week-old turkeys appeared to have a greater capacity to
form myotubes in vitro as measured by the percentage of total nuclei in

myotubes (fusion percentage) (Doumit et al., 1990). Although it is possible that

13

satellite cells may have lost an in vivo phenotype when cultured, these results

indicate that satellite cells from older animals retain the capacity to differentiate

' and fuse in vitro.

Although a number of studies demonstrate that indices of satellite cell
activity decline as a function of age, many of these studies used non-meat animal
species. Due to animal expenses as well as the prohibitive costs of radioactive
isotopes and animal disposal, studies examining satellite cell activity in species
used for meat production have been confined to the earliest stages of postnatal
growth. Therefore, characterization of satellite cell activity throughout postnatal
growth for meat animal species is required so that strategies can be developed
and employed to optimize satellite cell activity which will in turn impact DNA
accretion and ultimately muscle growth.

Age-related changes in satellite cell activity may be explained in part by
changes in the intercellular concentrations of growth factors. Stimulation of
satellite cell proliferation by homogenized muscle extracts was inversely
proportional to the age of rat from which the muscles were isolated (Mezzogiomo
et al., 1993). Furthermore, conditioned media from myotube cultures derived
from young rats stimulated myosin heavy chain (MHC) expression in satellite cell
cultures from 1 and 26-month-old rats. However, conditioned media from
myotube cultures of old mice failed to stimulate MHC expression in satellite cells
from either young or old animals. These data demonstrate that age-related
changes in satellite cell activity may be modulated in part by the paracrine

secretion of growth factors.

14

Effects of Growth Factors on Satellite Cell Activity
A. Hepatocyte Growth Factor (HGF)

In classical studies by Bischoff (1986b), crushed muscle extracts (CME)
were demonstrated to contain mitogenic factors that induce quiescent satellite
cells to enter the proliferative state. When cultures of single viable muscle fibers
from rat flexor digitorum brevis muscle were exposed to CME in basal medium,
quiescent satellite cells were induced to enter the cell cycle in vitro. Specificity
was confined to myogenic cells, as fibroblast cultures treated with CME did not
accumulate significant amounts of 3H-Thymidine. Direct injections of CME and
3H-Thymidine into the flexor digitorum brevis muscle of 1-week-old rat pups
demonstrated that satellite cell proliferation can be stimulated in vivo, causing
increases in total DNA content and myonuclei per fiber (Bischoff, 1986b).

Attempts to characterize the unknown mitogen in CME revealed that it had
a molecular mass greater than 30 kilodaltons and was heat and trypsin sensitive
(Bischoff, 1986b). In an effort to further characterize the mitogen, Chen et al.
(1992) introduced CME with saturating concentrations of FGF-2, insulin-like
growth factor-I (lGF-I), transferrin, platelet-derived growth factor (PDGF),
epidermal growth factor (EGF), adrenocorticotropic hormone (ACTH) and
macrophage colony-stimulating factor (M-CSF) to myogenic cultures isolated
from 5-day-old Swiss-Webster mice. Crushed muscle extract acted additively
with these growth factors to promote proliferation, indicating that the unknown

factor responsible for the mitogenicity of CME is distinct from those investigated

15

(Chen et al., 1992). In an attempt to further identify the factor(s) responsible for
its activity, CME was then separated into distinct mitogenic activities using
heparin affinity chromatography (Chen et al., 1994). Crushed muscle extract was
found to contain a protein that shares antigenicity with the BB isoforrn of PDGF,
as well as another protein that elutes from the column under identical conditions
as FGF-2. In addition, an unknown mitogenic component eluted from the column
at 0.9 M NaCl. This mitogen was shown to act additively with a combination of
saturating doses of FGF-2, lGF-l and EGF and also preferentially stimulated
proliferation in CZ myoblasts but not fibroblasts (Chen et al., 1994). More
recently, hepatocyte growth factor (HGF) was identified as the unknown
component of CME using western immunoblotting (T atsumi et al., 1998).
Furthermore, pre-incubation of CME with anti-HGF antibodies abolished the
mitogenic activity of CME.

Hepatocyte growth factor can also increase the rate of proliferation of
those satellite cells that have entered a proliferative state. Satellite cells isolated
and cultured from 9-month-old Sprague Dawley rats were shown to have a lag
phase leading to division of approximately 42 to 60 hrs. However, in the
presence of 10 nglml HGF, cells enter the cell cycle earlier and show a
significant increase in cell number as early as 24 hours in culture when
compared to control cultures. This increase in cell number is accompanied by
accelerated expression of cyclin D1 and PCNA, proteins required for progression
through the cell cycle. Messenger RNA for c-met, the HGF receptor, has been

detected in satellite cells from 0 to 72 hours in culture (Allen et al., 1995).

16

Injection of HGF into the tibialis anterior muscle of 1Z-month-old rats causes
quiescent satellite cells to enter the cell cycle and initiate DNA synthesis as
measured by BrdU incorporation (T atsumi et al., 1998). Hepatocyte Growth
Factor at 20 ng/ml has also been shown to increase the rate of DNA synthesis of
proliferating chicken satellite cells (Gal-Levi et al., 1998).

Hepatocyte growth factor also depresses differentiation of satellite cells.
Ectopic expression of chicken HGF by chicken satellite cells depressed
expression of MyoD and myogenin mRNA (Gal-Levi et al., 1998). MyoD and
Myogenin are muscle-specific transcription factors expressed early in the
differentiation program (Smith et al., 1994; Comelison and Wold, 1997).
Furthermore, ectopic expression of HGF suppressed expression of MHC protein
in these cells (Gal-Levi et al., 1998). Further studies indicated that depression of
transcription factor expression and differentiation may be a c-met mediated
event. When CZ mouse myoblasts are transfected with a constitutively activated
catalytic domain of c-met, expression of MyoD and myogenin mRNA as well as
MHC protein is depressed, indicating a depression of differentiation (Anastasi et
al., 1997). Since c-met mRNA is down-regulated preceding differentiation of
wild-type CZ myoblasts (Gal-Levi et al., 1998), it appears that differentiation may
occur through down-regulation of the c-met receptor.

Hepatocyte growth factor and its receptor c-met have been implicated in
modulating satellite cell activity in an autocrine action. Hepatocyte growth factor
is localized to the extracellular matrix surrounding rat muscle fibers, and the

associated satellite cells express c-met (Tatsumi et al., 1998). In regenerating

17

muscle, HGF appears to be released from the extracellular matrix and is
colocalized with immunostaining for c-met on the surface of satellite cells.
Hepatocyte growth factor mRNA has been detected in myotubes derived from
chicken satellite cells and CZ myoblasts (Gal-Levi et al., 1998) as well as in
proliferating CZ myoblasts (Anastasi et al., 1997). C2 cultures exposed to an
acid treatment followed by 0.5 M NaCl to remove HGF from the extracellular
matrix and dissociate receptor-ligand complexes results in a net decrease in 0-
met tyrosine phosphorylation. Application of C2 conditioned media to these
cultures restores phosphorylation of the receptor, indicating that HGF interacts
with the c-met receptor in an autocrine fashion (Anastasi et al., 1997).
Hepatocyte growth factor secretion by muscle appears to be
developmentally regulated. HGF mRNA has been detected in fractionated
muscle RNA isolated from 2, 4 and 10—day-old rats, but cannot be detected in
muscle RNA isolated from 35-day-old rats. However, HGF mRNA is reexpressed
in regenerating muscle and satellite cells of adult rats 3 to 4 days after ischemic
injury (Jennische et al., 1993). It is unknown if the developmental decline in
muscle HGF may be in part responsible for the observed age-related transition of

proliferating satellite cells to the quiescent state.

B. Fibroblast Growth Factor-2 (FGF-Z)
Fibroblast growth factor-2 has also been implicated in activating quiescent
satellite cells to enter a proliferative state. Cultures of rat muscle fibers treated

with FGF-Z or HGF exhibit increases in the number of PCNA+ satellite cells by

18

day 2 in vitro. However, PCNA labeling in HGF treated fiber cultures decreased
at day 4 in culture, yet FGF-2 treated cultures did not exhibit a decrease until day
5. These data indicated that FGF-Z, in addition to activating quiescent satellite,
is also capable of maintaining cells in a proliferative state (Yablonka-Reuveni et
al,1999)

Early studies by Gospodarowicz et al. (1976) demonstrated that the FGF
family of growth factors was capable of influencing myoblast proliferation and
differentiation. Using myoblasts isolated from bovine fetuses, FGFs purified from
either bovine brain or pituitary gland stimulated proliferation in vitro.

Furthermore, cultures maintained in the presence of FGF did not differentiate and
fuse to form multinucleated myotubes to the same extent as control cultures.
Both pituitary and brain derived FGFs have been reported to repress muscle CK
activity in the BCaH1 clonal mouse muscle cell line, indicating a depression of
differentiation and muscle-specific gene expression (Lathrop et al., 1985).
However, pituitary-derived FGF also caused an increase in proliferation in the
BC3H1 cell line, whereas brain FGF did not elicit a response. These data
demonstrate that FGFs are capable of stimulating proliferation and depressing
diffferentiation in myogenic cells.

Fibroblast growth factor-2 has been immunolocalized to the heparin-
containing basal lamina of mouse skeletal muscle fibers (DiMario et al., 1989),
yet conflicting data exist on the cellular origins of secreted FGFs. Rat primary
satellite cells have been shown to express acidic-FGF (FGF-1) protein, but not

basic-FGF (FGF-Z) (Groux-Muscatelli et al., 1990). However, FGF-Z appears to

19

be approximately 30-fold more potent than FGF-1 in stimulating proliferation of
the MM14 mouse myoblast cell line (Clegg et al., 1987). Both FGF-1 and FGF-2
mRNA have been detected in proliferating myoblasts but are absent from
differentiating cells of the MM14 cell line (Hannon et al., 1996). Furthermore,
transfection of these cells with FGF-1 and FGF-2 expression constructs mimics
the effects of exogenously applied FGFs, indicating that these cells are capable
of secreting functional FGFs. Although the origins of FGF-2 in vivo remain
unresolved, FGF-2 is a potent mitogen for satellite cell mediated skeletal muscle
growth.

Fibroblast growth factor-2 appears to act through maintenance of a
proliferative state, thereby depressing differentiation and muscle-specific gene
expression. Growing MM14 myoblasts become postmitotic, begin to express
muscle-specific genes, then fuse within 1Z-14 hours after FGF-2 withdrawal
(Clegg et al., 1987). Commitment to differentiation occurs in the G1 phase of the
cell cycle, with those cells in a post-G1 state completing the cell cycle before
commitment to terminal differentiation. Serum and FGF-2 are required to
maintain proliferation in this cell type. In the absence of serum, cultures can be
maintained in a post-mitotic state without commitment to differentiation. These
data indicate that repression of differentiation and stimulation of proliferation by
FGF-Z occurs through independent.

Developmental changes in FGF-Z levels as well as changes in the
mitogenic effect of FGF-2 on satellite cells have been investigated. FGF-Z

mRNA levels in skeletal muscle are initially high at 90 days of gestation in the pig

20

fetus, but decline until birth and remain constant to 180 days after birth (Peng et
al., 1997). Satellite cells isolated from 3-4-week-old rats exhibit specific, high
affinity binding of FGFZ to the fibroblast growth factor receptor-1 (FGFR-1) in
vitro as early as 18 hours post-plating, whereas cells from 9-12-month-old rats do
not exhibit binding until 42 hours post-plating (Johnson and Allen, 1993). These
data may in part explain the extended lag phase observed when satellite cells
are isolated and cultured from older rats. Yablonka-Reuveni et al., (1999)
demonstrated an increase in both the number of satellite cells (mitogen-activated
protein kinase positive cells (MAPK+)) as well as a number of proliferating
satellite cells (MAPK+/PCNA+) due to FGF—Z treatment of muscle fiber cultures
isolated from 3-week, 8-week and 10-month-old rats. Interestingly, The number
of MAPK+ and MAPK+/PCNA+ satellite cells were higher for 10-month-old rats
when compared to the younger rats. Furthermore, treatment of cultures with
FGF-2 caused a greater number of satellite cells to enter the myogenin-I-
compartment, indicating that differentiation was enhanced in this culture system.
It is possible that these conflicting results are a result of the continued
association of satellite cells with the native fiber, or secretion of growth factor(s)
by the fibers themselves. It is unknown if FGF-Z is responsible for regulating
age-related changes in satellite cell activity through activation of quiescent
satellite cells. Furthermore, the role of FGF-2 in maintaining the proliferative

state throughout growth remains to be elucidated.

21

C. Insulin-like Growth Factor-I (lGF-l)

Insulin-like growth factor-I is unique among growth factors because it is
able to elicit both mitogenic and myogenic responses, through interaction with
the same receptor (Insulin-like growth factor receptor-1 (IGFR-1)) (Florini et al.,
1996). Insulin-like growth factor-I increased proliferation of L6 myoblast cultures
and increased expression of c-fos mRNA and Cyclin D protein (Coolican et al.,
1997). In cultures of differentiating L6 myoblasts, lGF-l also increased CK
activity (Florini et al., 1997; Coolican et al., 1997), and up-regulated expression of
myogenin mRNA and protein (Florini et al., 1997). It appears that stimulation of
proliferation and differentiation occurs through activation of MAPK kinase and
phosphatidylinositol 3-kinase/p7056" pathways, respectively (Coolican et al.,
1997).

Interestingly, IGF-l appears to have synergistic effects on satellite cell
proliferation and differentiation when in the presence of FGF-2. When rat
satellite cells are exposed to a combination of FGF-2 and lGF-l, proliferation and
differentiation are synergistically enhanced (Allen and Boxhom, 1989). When
porcine myogenic clones were treated with FGF-2 and lGF-l, a synergistic
stimulation of proliferation was observed (Doumit et al., 1993). The mechanism
for this synergistic response is unknown. The potential interactions between
IGF-I and HGF on porcine satellite cell activity also remain to be established.

Insulin-like growth factor binding proteins (lGFBPs) are also capable of
modulating the response of satellite cells to IGF-l. Insulin-like growth factor

binding protein-4 is capable of depressing both proliferation and differentiation of

22

the L6 myoblast cell line. However, lGFBP-5 inhibits proliferation but enhances
differentiation of L6 myoblasts. It appears that the role of IGFBP-4 is mainly to
sequester excess lGFs, whereas expression of IGFBP-S‘elicits a dual response,
possibly due to its ability to associate with the cell membrane (Ewton et al.,

1 998).

The IGF-l regulatory system appears to be regulated in skeletal muscle
throughout growth. Muscle IGF-I mRNA levels are reported to be low at 30 days
of gestation in the pig fetus, increase between 44 and 59 days and plateau until
75 days, where levels rise again and peak at birth (Gerrard et al., 1997). In
agreement with these findings, Peng et al. (1996) reported that IGF-l mRNA
levels are initially high in pig fetuses from 90 days to 110 days of gestation, then
decline from birth to 180 days of age. Similarly, IGF receptor-1 (lGFR-1) mRNA
is also reported to be high at 110 days of gestation, then declines to basal levels
at 21 days after birth and remain constant throughout postnatal life. IGF binding
protein-3 (lGFBP-3) mRNA levels decline from high levels at 90 days of gestation
to basal levels at 21 days after birth and remain low to 180 days (Peng et al.,
1996). Insulin-like growth factor-I mRNA and protein have both been localized to
the developing muscle fibers (Gerrard et al., 1997). Taken together, these data
demonstrate that levels of muscle IGF-l, lGFR-1 and lGFBP-3 transcripts are
high in the pig fetus before birth, then decline throughout postnatal development.
The relationship between satellite cell activity and the developmental changes in

IGFI, lGFR-1 and IGFBPs are currently unresolved.

23

Several lines of evidence indicate that age-related changes in satellite cell
proliferation and differentiation are mediated through changes in the intercellular
levels of specific growth factors. In order to develop strategies to optimize
skeletal muscle growth in meat animal species, the mechanisms by which these
growth factors affect satellite cell activity need to be elucidated. Of the possible
candidates, HGF, FGF-2 and IGF are most likely the major mediators of skeletal
muscle DNA accretion. Collectively, these growth factors have the ability to
activate quiescent satellite cells, as well as stimulate proliferation and/or
differentiation. Interactions among these growth factors need to be elucidated.
Additionally, tissue levels of these growth factors and their influence on DNA
accretion and skeletal muscle growth in vivo remain to be established.

Based on the evidence presented above, I hypothesized that populations
of proliferating and differentiating satellite cells decline in vivo from 1 to 21 weeks
of age and that this decline limits DNA accretion and subsequent skeletal muscle

growth. The following objectives were designed to test this hypothesis:

Objective 1: Develop an immunocytochemical staining system to quantify in vivo
populations of proliferating and differentiating satellite cells.

Objective 2: Characterize the age-related changes in satellite cell proliferation
and differentiation from 1 to 21 weeks of age.

Objective 3: Relate changes in satellite cell activity to DNA accretion and

postnatal skeletal muscle growth.

24

CHAPTER 2
QUANTIFICATION OF SATELLITE CELL PROLIFERATION AND
DIFFERENTIATION IN GROWING PIGS FROM 1 TO 21 WEEKS OF AGE

Abstract
Postnatal muscle DNA accumulation occurs through proliferation of satellite cells
followed by differentiation and fusion of these cells with existing muscle fibers.
Furthermore, muscle DNA accumulation is proportional to fiber diameter or
growth. Therefore, characterization of the age-related changes in satellite cell
activity is essential to the understanding of skeletal muscle growth. Satellite cells
were isolated from the semitendinosus (ST) muscle of pigs at 1, 7, 14, and 21
weeks of age (4 animals/age group). Satellite cells were separated from cellular
debris using Percoll gradient centrifugation, and adsorbed to glass coverslips for
fluorescent immunostaining. Positive staining for neural cell adhesion molecule
(NCAM) distinguished satellite cells from non-myogenic cells. Proliferating cell
nuclear antigen (PCNA) and myogenin were used as markers for quantification of
proliferating and differentiating satellite cells, respectively. DNA and protein
concentrations of the ST and Iongissimus dorsi (LD) were determined and used
to calculate total protein and DNA accretion as well as the proteinzDNA ratio.
Accretion of DNA and protein closely paralleled increases in muscle weights of
the ST and LD. Furthermore, the protein to DNA ratio remained constant in the
ST and LD from 1 to 7 weeks of age, then increased for both muscles from 7 to

21 weeks of age. The proportion of satellite cells (NCAM+ cells) was highest in

25

isolates from 1-week-old pigs, yet the proportion of proliferating satellite cells
(NCAM+/PCNA+ cells) declined only slightly with age. Furthermore, a high
degree of proliferation (>75% PCNA positive nuclei) wasimaintained to 21 weeks
of age. The percentage of differentiating satellite cells decreased from 30% at 1
week to 14% at 7 weeks of age and remained at constant levels thereafter.
These data indicate that a high percentage of satellite cells remain proliferative
throughout postnatal growth in pigs from 1 to 21 week of age. Differentiation

may be a critical step in the regulation of postnatal skeletal muscle hypertrophy.

Introduction

Postnatal skeletal muscle growth in the pig occurs through hypertrophy of
existing muscle fibers, since the number of muscle fibers is determined prenatally
and remains constant throughout postnatal life (Stickland et al., 1975; Wrgmore
and Stickland, 1982, 1983). Muscle fiber hypertrophy is associated with an
increased DNA content, yet myonuclei do not retain the ability to synthesize DNA
(Stockdale and Holtzer, 1961). Satellite cells, first discovered by Alexander
Mauro in 1961, are the sole postnatal source of DNA contributed to growing
muscle fibers. DNA accretion occurs through proliferation of satellite cells
followed by differentiation and fusion with existing muscle fibers (Moss and
LeBlond, 1971).

It is generally accepted that the proportion of satellite cells and
proliferating satellite cells declines with age. Campion et al. (1981) observed a

10% decrease in the proportion of nuclei classified as satellite cells in histological

26

sections of the peroneus Iongus and sartorius muscles from 1 to 64-week-old
pigs. However, these data suggest that the absolute number of satellite cells in
these muscles may actually increase from 1 to 32 weeks of age. Increases in the
absolute number of satellite cells throughout postnatal growth have also been
reported for the semimembranosus muscle of Japanese quail (Campion et al.,
1982) and the soleus muscle of the rat (Gibson and Schultz, 1983). Mulvaney et
al. (1988) demonstrated a decrease in satellite cell proliferation (based on total
myonuclei) as measured by incorporation of 3H-thymidine into satellite cells of the
triceps brachii muscle of growing pigs 1 to 21 days of age. Other investigators
have demonstrated similar declines in both the proportion of satellite cells as well
as the percentage of proliferating satellite cells in the later stages of growth in
laboratory animals (Allbrook et al., 1971; Cardasis and Cooper, 1975; Schultz
and Lipton, 1982). The age-related changes in satellite cell proliferation
throughout the rapid growth phase of porcine skeletal muscle have not been
characterized.

The rate of differentiation may be a potentially important modulator of
muscle growth as it regulates both the accretion of DNA into muscle fibers as
well as the number of satellite cells that remain capable of proliferation. Quinn et
al. (1990) demonstrated that embryonic myoblasts isolated from fetal calves with
the double-muscling phenotype exhibited a delay in differentiation compared to
myoblasts isolated from normal fetuses. This delay resulted in an enlarged
population of myoblasts resulting in an increased production of fused myotubes

in vitro. Coutinho et al. (1993) observed a similar delay in formation of the

27

brachial somites in quail embryos selected for rapid muscle growth. These data
indicate that the rate of differentiation may be a critical event in muscle
' development. To date, the age—related changes in satellite cell differentiation
during postnatal skeletal muscle growth have not been characterized.
Characterization of the age-related changes in satellite cell proliferation
and differentiation will provide insight into the cellular mechanisms governing
skeletal muscle hypertrophy. This information will be useful for development of
strategies to increase DNA accretion and skeletal muscle growth. Such
advances will also serve to increase the efficiency of lean tissue accretion

throughout postnatal growth.

Materials and Methods

A. Animals

Duroc sires were mated to F1 Yorkshire x Landrace sows and F2 progeny
were raised at the Michigan State University Swine Farm. Male piglets were
castrated at 1 day of age. F2 barrows and gilts at 1, 7, 14, and 21 weeks of age
(4 animals per age group) were transported to the Michigan State University
Meat Laboratory and slaughtered following procedures outlined in the Code of

Federal Regulations for Humane Slaughter of Livestock (Sections 3131-3139).

B. Satellite Cell Isolation
Satellite cells were isolated from the right hindlimb muscles of growing

pigs as described by Doumit and Merkel (1992). Due to low muscle mass of 1-

28

week-old pigs, satellite cells were isolated from both the semimembranosus and
semitendinosus muscles, while only the semitendinosus was used from 7, 14,
and 21-week-old pigs. Briefly, muscles were removed from the right hindlimb,
trimmed, weighed and rinsed in cold phosphate buffered saline pH=7.4 (PBS). In
a laminar flow tissue culture hood, muscle was cut into ~2 cm3 sections and
visible connective tissue was removed. Sections were then washed in cold
Minimum Essential Medium (MEM) pH=7.1 (11900-024, Gibco BRL, Grand
Island NY) and ground in an aseptically prepared meat grinder. Thirty grams of
muscle tissue were digested in PBS containing 0.8 mg/ml Pronase E (P-8811,
lot#88H1351, 3.9 units/mg, Sigma Chemical Co., St Louis M0) for 50 minutes at
37°C. Samples were vortexed at 10-minute intervals during enzymatic digestion.
Digestions were performed at a ratio of 40% tissue to 60% protease solution
(w/v). After digestion, samples were separated from the protease solution by
centrifugation at 1000 xg followed by washing of the pellets with PBS. Satellite
cells were separated from debris by differential centrifugation at 100 xg. Satellite
cells were collected by centrifugation at 1000 xg and pellets were re-suspended
in MEM supplemented with 20% Fetal Bovine Serum (FBS)(F-Z442,
Lot#76H4647, Sigma Chemical Co., St Louis M0), 0.5% Antibiotic/Antimycotic
and 0.1% Gentamicin (A-5955 and G-1397, Sigma Chemical Co., St LoUis MO).

Cell suspensions were filtered through ~500 pm and double layered ~53 um

Nitex cloth (3-500-49 and 3—53-41, Sefar America, Kansas City MO). Dimethyl

sulfoxide (#9224-01, J.T. Baker, Phillipsburg NJ) was added to the cell

29

suspension to 10% of the total volume and aliquots were frozen overnight in

styrofoam containers at -80°C and stored in liquid nitrogen until use.

C. Tissue Collection

Samples were taken from the left semitendinosus (ST) and right
Iongissimus dorsi (LD) muscles for determination of DNA and protein
concentrations. The ST was excised completely, trimmed of connective tissue,
weighed and samples were collected from the belly of the muscle. For 1 and 7-
week-old pigs, the LD was removed quantitatively from the cranial end of the
ilium to its termination in the shoulder then weighed. Longissmus dorsi samples
were taken from the region of the muscle adjacent to the 10‘" rib. In 14 and 21-
week-old pigs, samples were prepared from a 25-100mm thick section that was
excised immediately caudal to the 10th rib. The remainder of the LD was
removed quantitatively as described for 1 and 7-week-old pigs to determine total
muscle weight.

For histology studies, 5x5x10 mm samples were excised with the long axis
of the sample running parallel to the muscle fibers. Segments were coated in
Tissue-Tek O.C.T. Compound (4583, Sakura Finetek U.S.A. Torrance CA) then
frozen in Z-methylbutane (#32,040-4, Aldrich Chemical Company, Milwaukee,
WI) that had been cooled in liquid nitrogen vapors. The remainder of the muscle
section was cut into 1-2 cm3 sections, frozen in liquid nitrogen, and stored at

-80°C.

30

At exsanguination, 100 ml of blood was collected for serum isolation.
Blood was allowed to clot at room temperature for 2 hours, then stored at 4°C
overnight. Blood was centrifuged at 2000 xg for 30 minutes at 4°C and serum
was collected. Serum was centrifuged again, transferred to a new tube and
frozen at -80°C.

Representative samples of liver were also taken for further analysis. Liver
samples were cut into 1-2 cm3 sections, frozen in liquid nitrogen and stored at

-80°C.

D. DNA Assay

The DNA contents of the left semitendinosus and right Iongissimus dorsi
muscles were determined using the procedure of Labarca and Paigen (1980).
Briefly, 1.0 g of muscle was homogenized (4, 30-second bursts) in 25 volumes
(wlw) of cold extraction buffer (10 mM Tris, 5 mM EDTA, pH=8.0) using a
Polytron homogenizer (Brinkmann, Westbury NY). Triplicate 100 pl aliquots of
muscle homogenate were mixed with 3 ml of DNA assay buffer (0.05 M
NaH2PO4oH20, Z M NaCl, 2 mM EDTA, pH=7.4 containing 1 jig/ml Hoechst
33258 reagent (B-2883, Sigma Chemical Co., St. Louis MO) in methacrylate
cuvets (14-38621, Fisher Scientific, Itasca IL) pre-selected for optical uniformity.
A serial dilution (100 to 3.125 pglml) of Calf Thymus DNA (D-0805, Sigma
Chemical Co., St. Louis MO) in extraction buffer served as a standard and was

treated in an identical fashion to the samples. Samples and standards were

31

incubated in the dark for 15 minutes then read with a DynaQuant fluorometer

(Hoefer Phannacia Biotechnology Inc., San Francisco CA).

E. Biuret Protein Assay

Determination of protein content of the left semitendinosus and right
Iongissimus dorsi muscles was accomplished using aliquots of the muscle
homogenate following a procedure outlined Gornall et al. (1948) and modified by
Robson et al. (1968). Briefly, triplicate 250 pl aliquots were treated with 750 pl of
1 N NaOH, vortexed and incubated at 37°C for 3-4 hours. A serial dilution of
bovine serum albumin (A-2153, Sigma Chemical Co., St. Louis MO) in 25%
extraction buffer, 75% 1 N NaOH served as a standard and was treated in an
identical fashion to the samples. Four milliliters of biuret reagent (6 mM
CuSO4-5H20, 20 mM KNaC4H40604H2O, 750 mM NaOH) were added to samples
and standards and incubated in the dark for 30 minutes. Two hundred and fifty
microliters of sample and standards were placed into an lmmulon 1 96 well

microtiter plate (#14 24578, Fisher Scientific, Itasca IL) and read at 540nm.

F. Culture of 5.1H11 and FSD Hybridoma Cell Lines

The 5.1 H11 cell line was developed by Drs. Helen Blau and Frank Walsh
at Stanford University School of Medicine, Stanford CA. The F50 cell line was
developed by Dr. Woodring Wright at the University of Texas Southwestern
Medical Center, Dallas TX. The cell lines were obtained from the Developmental

Studies Hybridoma Bank maintained by the University of Iowa, Department of

32

Biological Sciences, Iowa City, IA 52242 under contract NO1-HD-7-3263 from
the National Institute of Child Health and Human Development (NICHD).

5.1H11 hybridoma cells were grown in 75 cm2 tissue culture flasks (#3376,
Coming Incorporated, Corning NY) in Dulbecco’s Modified Eagle Medium with 25
mM HEPES (#23700-040, Gibco BRL, Grand Island NY) containing 10% FBS,
0.1 mM Non-Essential Amino Acids (#11140-050, Gibco BRL, Grand Island NY),
1 mM sodium pyruvate (S-8636, Sigma Chemical Co., St. Louis MO), 2 mM L-
glutamine (G-5763, Sigma Chemical Co., St. Louis MO) and 0.5%
Antibiotic/Antimycotic. FSD hybridoma cells were grown in 75 cm2 tissue culture
flasks in RPMI medium 1640 (#31800-022, Gibco BRL, Grand Island NY)
containing 10% FBS and 0.1% Gentamicin. All cultures were maintained in a
37°C humidified incubator containing 95% air and 5% CO2. Frozen cell
suspensions were thawed in a warm water bath and re-suspended in 10 ml of
media to dilute freezing medium and centrifuged at 300 xg for 2 minutes. The
cell pellet was then re-suspended and plated at 2.5x10‘5 cells/75 cm2 flask in 20
ml of medium (125,000 cells/ml). Cells were supplied with new medium every 48
hours at a ratio of 1:4 (existing cell suspension to fresh media) and cell
suspensions were transferred into new flasks when necessary. Feeding was
continUed until the desired volume of supernatant was attained. Cultures were
then left undisturbed for 12-15 days. Cultures were harvested, pooled and
centrifuged at 2000 xg for 15 minutes to pellet cells. The clarified supernatant
was then collected and stored in aliquots at -20°C until use. Thawed aliquots

were stored at 4°C.

33

G. Satellite Cell Enrichment and Adsorption to Coverslips

Porcine satellite cells were separated from debris using Percoll gradient
centrifugation as described by Yablonka-Reuveni (1989). Briefly, a 90% Percoll
solution (P-1644, Sigma Chemical Co., St Louis MO) was prepared in 10x MEM.
Percoll solutions (60% and 20%) were prepared in 1x MEM as dilutions of the
90% stock. Two milliliters of 60% Percoll was added to a 15 ml Corex tube that
had been pre-treated for 2 hours with either FBS or horse serum (H-1270, Sigma
Chemical Co., St. Louis MO). Eight milliliters of 20% Percoll solution was then
layered upon the 60% solution in the tube. One vial of primary muscle cell
suspension was thawed in a warm water bath and transferred to a 15 ml conical
tube. Two milliliters of MEM were used to wash the vial(s) and was added to the
cell suspension. Diluted cell suspensions were loaded onto the gradients and
centrifuged at 15,000 xg for 5 minutes at 4°C with the brakes off. The interface
between the 20% and 60% Percoll solutions was recovered and diluted with 20
volumes of MEM. The diluted interface was then centrifuged at 300 xg for 15
minutes to pellet cells. The supernatant was then decanted and the cell pellet
was re-suspended in the remaining volume (0.5-1 ml) of MEM. The cell
suspension was transferred to a microcentrifuge tube and centrifuged at 1400 xg
for 5 minutes. The supernatant was aspirated and the cell pellet was re-
suspended in 200 pl of MEM. One hundred microliters of the resulting cell
suspension was allowed to adsorb to 12 mm glass coverslips (12-545-82, Fisher

Scientific, Itasca IL) for 45 minutes. For each satellite cell isolate, cells were

34

adsorbed onto two coverslips for staining for PCNA and Myogenin, respectively.
Coverslips with adsorbed cells were washed once with PBS+2% Goat Serum (G-

6767, Sigma Chemical Co., St Louis MO) before blocking and staining.

H. Immunostaining for Neural Cell Adhesion Molecule (NCAM)

All washes and antibody incubations were performed in a volume of 0.5
ml. Porcine satellite cells were incubated in PBS with 2% goat serum (blocking
solution) for 5-10 minutes to block non-specific binding of antibodies. Cells were
then incubated in undiluted 5.1H11 hybridoma supernatant (containing anti-
NCAM antibodies) for 30 minutes (see Table 2.1. for description of antigen),
followed by a series of three washes in blocking solution. Incubation of 1 pg/ml
non-specific mouse lgG (l-5381, Sigma Chemical Co, St Louis MO) in the place
of 5.1H11 hybridoma supernatant served as a negative control and subsequent
steps were performed in an identical fashion. Cells were incubated for 30
minutes in blocking solution with 0.4 pg/ml biotinylated goat, anti-mouse lgG1
(#M32015, Caltag Laboratories, Burlingame CA), and the washing series was
repeated. To detect primary antibody binding, cells were incubated for 30
minutes in the dark with ExtrAvidin conjugated to tetramethylrhodamine
isothiocyanate (TRITC) (E-3011, Sigma Chemical Co., St Louis MO) diluted
1:200 in blocking solution. Cells were washed once in blocking solution then
fixed with 1% formalin in PBS (#2106-01, J.T. Baker, Phillipsburg NJ) for 10

minutes, followed by a 10-minute exposure to -20°C methanol (#9070-03, J.T.

35

Baker, Phillipsburg NJ). The washing series was repeated and cells were

stained for either the presence of PCNA or Myogenin.

I. Immunostaining for Proliferating Cell Nuclear Antigen (PCNA)

Porcine satellite cells that had been stained for the presence of NCAM
(described above) were incubated in 1 pglml anti-PCNA monoclonal antibody
(#1486 772, Boehringer Mannheim, Indianapolis IN) in blocking solution for 1-2
hours (see Table 2.1 for description of antigen). Incubation of 1 pglml non-
specific mouse lgG in the place of anti-PCNA antibody served as a negative
control and subsequent steps were performed in an identical fashion. Cells were
washed again and incubated for 30 minutes in 0.8 pglml goat, anti-mouse lgG2a
conjugated to fluorescein isothiocyanate (FITC) (#M32301, Caltag Laboratories,
Burlingame CA) in blocking solution. The washing series was repeated and
coverslips were mounted onto microscope slides with 2.5 pl of VectaShield
mounting medium containing 4',6-diamidino-Z-phenylindole (DAPI) to
counterstain DNA (H-1200, Vector Laboratories, Burlingame CA). Coverslips
were sealed with nail polish and cells were viewed using a Leica DMLB
fluorescent microscope. A minimum of 600 cells were evaluated at 400x in
random or sequential fields of view. For each field of view, total cell number,
total proliferating (PCNA+) cells, total satellite cells (NCAM+), and proliferating
satellite cells (NCAM+/PCNA+) were enumerated by two independent evaluators
blinded to the identity of the sample. The number of non-myogenic (NCAM-) and

proliferating non-myogenic (NCAM-IPCNA+) cells were calculated by difference.

36

J. Immunostaining for Myogenin

Porcine satellite cells that had been stained for the presence of NCAM
(described above) were incubated in undiluted F5D hybridoma supernatant
(containing anti-myogenin antibodies) for 1-2 hours (see Table 2.1 for description
of antigen). Incubation of 1 pglml non-specific mouse lgG in the place of F5D
hybridoma supernatant served as a negative control and subsequent steps were
performed in an identical fashion. Cells were then incubated with 1:100 goat,
anti-mouse lgG conjugated to F ITC (F-0257, Sigma Chemical Co., St Louis MO)
in blocking solution for 30 minutes. The washing series was repeated and cells
were mounted onto microscope slides with 2.5 pl of VectaShield mounting
medium containing DAPI counterstain. The slides were sealed with nail polish
and viewed using a Leica DMLB fluorescent microscope. A minimum of 700
cells were evaluated using the 400x objective in random or sequential fields of
view. For each field of view, total cell number, total satellite cells (NCAM+), and
total myogenin positive cells (Myog+) were enumerated by two independent

evaluators blinded to the identity of the sample.

K. Statistical Analysis
Data sets were analyzed with Statistical Analysis Software (SAS Institute
Inc., Cary NC). Percentage data were converted to a decimal value and

transformed as the inverse sin of the square root. A one-way Analysis of

37

Variance (ANOVA) and Bonferroni multiple comparisons test was used to

compare treatment means with a Type I error rate of 5%.

Results

A. Live Weights and Muscle Weights

Live weight increased throughout the experimental period (3.4 kg at 1
week to 89.2 kg at 21 weeks); (Figure 2.1.). Semitendinosus (ST) muscle weight
increased in proportion to body weight with the most rapid increase in cumulative
weight gain occurring between 14 and 21 weeks of age (9.7 g at 1 to 463.7 g at
21 weeks (Figures 2.2.). Longissimus dorsi (LD) muscle weight also increased
throughout the experimental period (45.0 g at 1 week to 1450.0 9 at 21 weeks)
with the fastest rate of growth occurring between 7 and 14 weeks of age. At 1 to

7 and 14 to 21 weeks of age comparatively slower rates of growth were observed

in the LD (Figure 2.3.).

B. DNA and Protein Contents

Total DNA and protein contents of the ST and LD muscles are presented
with the changes in muscle weight in Figures 2.2. and 2.3., respectively.
Increases in total DNA and protein of both muscles closely parallel increases in
muscle weight. Figure 2.4. shows the calculated number of nuclei accumulated
per day for 1 to 7, 7 to 14, and 14 to 21 weeks of age. Myonuclei were assumed
to have approximately 7.4 pg of DNA (Doumit et al., 1993) with the majority of

DNA present in muscle belonging to myonuclei. Based on Figure 2.4., 18.6%,

38

36% and 45.3% of myonuclei accumulated within 1 to 7, 7 to 14, and 14 to 21
weeks of age, respectively.

The protein to DNA ratios for the ST and LD are presented in Figure 2.5.
From 1 to 7 weeks of age, protein accumulation in the ST and LD occurred
proportionately to DNA accretion. However, from 7 to 21 weeks of age, protein

accumulated at a greater rate than did DNA in both the ST and LD.

C. Percentage of Satellite Cells (NCAM+ Cells)

Panel B of Figure 2.6. demonstrate representative staining for the
presence of NCAM. Closed and open arrows indicate positive and negative
staining, respectively. Panel B of Figure 2.9. depicts representative non-specific
staining for NCAM. NCAM immunostaining is characterized by intense TRITC
staining in a punctate pattern that is confined to the surface of satellite cells. The
percentage of NCAM+ cells declined from 57.9% to 33.8% from 1 to 7 weeks of
age and remained constant thereafter (P < 0.05); (Figure 2.10.). The
percentages of satellite cells in coverslips stained for the quantification of
differentiating satellite cells (Myogenin+ cells) were similar to the above data
(data not shown). Conversely, the percentage of non-myogenic (NCAM- cells)

increased from 42.1% in 1-week-old pigs to 66.2% in 7-week-old pigs (P < 0.05).

D. Percentage of Proliferating Satellite Cells (NCAM+/PCNA-I- Cells)

Panel B of Figure 2.7. depicts representative staining for the presence of

PCNA. Closed and open arrows indicate positive and negative staining,

39

respectively. Panel D of Figure 2.9. demonstrates representative non-specific
staining for PCNA. PCNA immunostaining is characterized by intense FITC
staining confined to the nuclei of cells. Although there was a decline in the
percentage of proliferating satellite cells between 1 and 14 weeks of age (P

<0. 05), no other differences were detected (Figure 2.11.). A high proportion
(79.4%) of satellite cells isolated from the ST muscle of 21-week-old pigs remain

in a proliferative (PCNA+) state.

E. Percentage of Proliferating Non-Myogenic Cells (NCAM-IPCNA+ Cells)
The percentage of proliferating non-myogenic cells increased from 38.9%
in 1-week-old pigs to 74.1% in 7-week-old pigs (P < 0.05); (Figure 2.12.). No
differences in the percentage of proliferating non-myogenic cells were detected

at 7, 14, or 21 weeks of age.

F. Percentage of Differentiating Satellite Cells

Panel B of Figure 2.8. demonstrates representative immunostaining for
myogenin. Closed and open arrows indicate positive and negative staining,
respectively. Panel F of Figure 2.9. depicts representative non-specific
immunostaining for myogenin. Myogenin staining is characterized by intense
FITC staining that is confined to the nuclei of satellite cells. The percentage of
differentiating satellite cells were 30.7% in 1-week-old pigs, but decreased to

14.4% in 7-week-old pigs (P <0.05) (Figure 2.13.). The percentage of

40

differentiating satellite cells continued to decline numerically from 7 to 21 weeks

ofage.

Discussion

Powell and Aberle (1975) demonstrated that in lines of swine differing in
muscularity, increased muscle mass is associated with an increased DNA
content. Therefore, it is generally accepted that DNA accretion is essential to
muscle hypertrophy and growth. Since those nuclei incorporated into muscle
fibers are incapable of DNA synthesis (Stockdale and Holtzer, 1961), DNA
accretion must occur by other cellular mechanisms. Satellite cells facilitate DNA
accretion through proliferation, followed by differentiation and fusion with existing
muscle fibers, thereby increasing the capacity of the muscle fiber to synthesize
myofibrillar protein (Moss and Leblond, 1971; Cheek et al., 1970, 1971). The
objective of the present study was to characterize the age-related changes in
populations of proliferating and differentiating satellite cells in growing pigs from 1
to 21 weeks of age.

It appears that the ST muscle is in a rapid state of growth from 1 to 21
weeks of age and is accommodated by concurrent increases in both total DNA
and protein (Figure 2.2). Changes in ST muscle growth closely parallelled
changes in total live weight (Figure 2.1.) from 1 to 21 weeks of age, which makes
the ST a suitable model in which to study skeletal muscle growth (Dr. Robert
Merkel, personal communication). As in the case of the ST muscle, DNA and

protein contents of the LD muscle also closely paralleled changes in total muscle

41

weight, indicating a similar association of muscle growth to DNA and protein
accretion (Figure 2.3.). However, the largest gain in LD muscle weight occurred
’ from 7 to 14 weeks of age with relatively slower rates of gain at the early and late
phases of growth. The cellular mechanisms contributing to the observed
differences in ST and LD muscle growth from 1 to 21 weeks of age are unknown.

Regardless of the observed differences in muscle growth of the ST and
LD, both muscles exhibited similar changes in the protein to DNA ratio from 1 to
21 weeks of age (Figure 2.5.). It appeared that from 1 to 7 weeks of age, ST and
LD DNA and protein accumulate in a proportional manner. However, at the later
stages of growth (7 to 21 weeks) protein accumulated at a greater rate than
DNA. This indicates that an individual skeletal muscle DNA unit in these muscles
are capable of accommodating a larger protein pool at the later phases of the
experimental period.

The proportion of satellite cells as a percentage of the total cell population
isolated from the ST muscle declines with age (Figure 2.10.). Other studies for
the pig (Campion, 1981), the mouse (Cardasis and Cooper, 1975) and the rat
(Allbrook et al., 1971) demonstrated age-related declines in the proportion of
nuclei classified as satellite cells. The current study provides the first evidence to
indicate that there is an apparent increase in the proportion of non-myogenic
(NCAM-) cells to satellite cells (NCAM+) in muscle of growing pigs from 1 to 7
weeks of age.

A number of explanations can account for the observed increase in

proportion of non-myogenic cells to satellite cells. A relatively high level of

42

differentiation at 1 week of age (Figure 2.13.) indicates that a greater proportion
of satellite cells are committed to differentiation and may not be extracted from
the tissue using the present isolation method. Therefore, as a percentage of total
cells isolated from the ST muscle, the proportion of satellite cells would decrease
relative to non-myogenic cells. It is possible that changes in the levels of one or
more mitogens in muscle may influence the proportion of satellite cells to non-
myogenic cells. Vandenburgh et al. (1984) demonstrated that a 16 to 22-hour
exposure of embryonic chicken muscle cultures to muscle extracts from 86-day-
old mice increased the proportion of fibroblastic cells when compared to extracts
from younger (Z8-day-old) mice. It is interesting to note that the percentage of
proliferating non-myogenic cells (NCAM-IPCNA+) also increased at the later time
points examined in this study (Figure 2.12.). Mitogens released from crushed
extracts of rat muscle have been shown to stimulate the proliferation of satellite
cells but not muscle derived fibroblasts (Bischoff, 1986b; Chen et al., 1994). The
active mitogen in crushed muscle extract, later determined to be hepatocyte
growth factor (HGF) (T atsumi et al., 1998), has been shown to be
developmentally regulated in skeletal muscle (Jennische et al., 1993). It is
possible that levels of HGF decrease throughout growth of porcine skeletal
muscle or satellite cells become unresponsive to HGF, thereby altering the
proportion of myogenic to non-myogenic cells. A number of other growth factors
may be capable of influencing the proportions of satellite cells to non-myogenic
cells and it is the relative levels of these growth factors in muscle that may

govern the in vivo proportions of satellite cells to non-myogenic cells.

43

It is generally accepted that at birth a high proportion of satellite cells exist
in a proliferative state and as an animal ages this proportion decreases as
satellite cells enter a quiescent state (reviewed by Allen et al., 1979). However,
the present study indicates that a relatively modest decline in satellite cell
proliferation occurs in the ST muscle of growing pigs (see Figure 2.11.).
Furthermore, a high proportion (greater than 75%) of satellite cells remain
proliferative at 21 weeks of age. Based on changes in ST muscle weight as well
as the rates of DNA and protein accretion, it is apparent that the ST muscle is
rapidly growing from 1 to 21 weeks of age (Figures 2.2.) Therefore, a high index
of satellite cell proliferation is not unexpected.

A number of studies have reported declines in satellite cell proliferation at
earlier ages in the pig (Mulvaney et al., 1988) and during growth in the turkey
(Mozdziak et al., 1994). However, these studies report a decline in satellite cell
proliferation (utilizing 3H-Thymidine or BrdU to label proliferating cells) as a
percentage of total muscle nuclei (which includes both incorporated nuclei and
satellite cell nuclei). Since the number of myonuclei per muscle fiber increases
throughout postnatal growth (Moss and Leblond, 1971), the percentage of
muscle nuclei that are satellite cells would be expected to decrease.
Consequently, the DNA labeling index would decline when expressed relative to
total muscle nuclei. The findings of the current study are in general agreement
with those of Schultz (1996), who reported that 80% of satellite cells from 30-day-
old growing rats proliferate with a 32-hour cell cycle as determined by dual-

labeling experiments utilizing 3H-Thymidine and BrdU. The remaining 20%

proliferate at a much slower rate, possibly entering a quiescent state between
divisions. Although the data of Schultz (1996) do not provide information about
changes in satellite cell cycle time at multiple time-points throughout growth, they
are in agreement with the high proportion of proliferating satellite cells reported in
this study examining rapidly growing pigs.

Proliferating cell nuclear antigen (PCNA) is undetectable in quiescent cells
via immunofluorescence when methanol is used as a fixative, and is up-regulated
in the G1 phase of the cell cycle, reaches a peak the S phase then declines
during G2 (Morris and Mathews, 1989; Bravo and MacdonaId-Bravo, 1987b). A
number of other studies have employed immunostaining against PCNA to
quantify the index of proliferation for satellite cells in vitro (Johnson and Allen,
1993; Yablonka-Reuveni and Rivera, 1994; Yablonka-Reuveni et al., 1999).
Although is it unknown to what extent porcine satellite cells express detectable
levels of PCNA throughout the cell cycle, it appears that PCNA expression is
correlated to DNA synthesis and is well suited for use as an index of proliferation.

Although not quantified in these studies, some heterogeneous PCNA
staining was observed with increasing age. Proliferating cell nuclear antigen
staining (PCNA) of 1-week-old pig satellite cells appeared to be fairly
homogeneous and intense whereas cells isolated from 21-week-old pigs
contained populations of PCNA positive cells with both intense and less intense
staining (data not shown). This observation, taken together with data reported by
Schultz (1996), indicate that changes may also occur in the proportion of porcine

satellite cells that progress through the cell cycle at different rates from 1 to 21

45

weeks of age. More work needs to be done to determine the nature of this
phenomenon and the impact on skeletal muscle growth.

To my knowledge, no information exists on the age-related changes in in
vivo satellite cell differentiation from birth to maturity in the growing pig. Data
reported in this thesis demonstrate that approximately 30% of satellite cells are
myogenin positive at 1 week of age and this percentage declines to
approximately 8% at 21 weeks of age (Figure 2.13.). It is interesting to note that
the majority of DNA accumulates from 7 to 21 weeks of age, a time at which the
low levels of satellite cell differentiation exist (Figure 2.4.). It is possible that the
high index of proliferation observed within the experimental period serves to
increase the absolute number of satellite cells within the muscle. Early studies
demonstrated an increase in the absolute number of satellite cells throughout
postnatal growth in the peroneus Iongus and sartorius muscles of the pig
(Campion et al., 1981), the semimembranosus muscle of Japanese quail
(Campion et al., 1982) and the soleus muscle in the rat (Gibson and Schultz,
1983). More recent studies by Yablonka-Reuveni et al. (1999) demonstrate that
a larger population of satellite cells (MAPK+ cell) exist in muscle fiber cultures
isolated from 10-month-old rats at plating compared to cultures from younger
rats. Thus, it appears that a large population of satellite cells may exist in the
muscle of older animals. In this case, although the rate of satellite cell
differentiation declines within the experimental period, the absolute number of
satellite cells that incorporate into the muscle fibers may remain high due to a

larger population of satellite cells. In addition, the decrease in the rate of

46

differentiation may also serve to maintain the population satellite cells capable of
proliferation (i.e. those that have not entered the differentiation program and have
become post-mitotic) causing a further increase in the absolute numbers of
satellite cells. Thus, it is possible to maintain a high level of incorporation of
nuclei into a muscle fiber even if a decrease in the rate of satellite cell
differentiation is observed.

Myogenin has been shown to be up-regulated in mononucleated
myogenic cells relatively early in the differentiation program and remain high until
fusion of the cell into a syncytial myotube or muscle fiber (Andres and Walsh,
1996). However, myogenin positive satellite cells retain the capability to
synthesize DNA and it is not until later in the differentiation program that these
cells become post-mitotic and begin to exhibit the contractile phenotype. At this
point it is unclear if satellite cells are capable of undergoing multiple rounds of
proliferation after becoming positive for myogenin or if differentiation proceeds
immediately after completing the current round of proliferation. However, since
myogenin expression indicates a commitment to terminal differentiation, this
marker provides a relative indicator of the proportion of cells committed to fusion.

At this time it is impossible to rule out programmed cell death or apoptosis
as a pessible fate of satellite cells quantified in this study. Apoptosis has been
observed in satellite cell lines in vitro in response to serum starvation or
staurosporine treatment (Mampuru et al., 1996; Maglara et al., 1998; McArdle et
al., 1999). However, it is unknown if satellite cells or incorporated myonuclei

undergo significant amounts of apoptosis throughout postnatal growth. A

47

number of recent studies have determined that apoptosis also exists as a
possible fate for incorporated myonuclei during development or in response to
damage, denervation and disease (Borisov and Carlson, 2000; reviewed by
Sandri and Carraro, 1999). Future work in this area will determine the role of
programmed cell death or apoptosis in DNA accretion and skeletal muscle
growth throughout postnatal growth. Apoptosis may be a mechanism employed
to control the size of a satellite cell population containing a high proportion of
proliferating and a decreasing proportion of differentiating cells as observed in
this study.

It is evident from the above study that satellite cells from the
semitendinosus muscle of growing pigs retain a high level of proliferation and
exhibit an age-related decline in satellite cell differentiation throughout postnatal
growth. Therefore, it appears that the differentiation and fusion may be a critical
step in regulation of skeletal muscle hypertrophy. Future studies should
determine the mechanisms by which satellite cell differentiation is regulated in
vivo in order to develop efficient growth-promoting agents for the optimization of
DNA accretion. Furthermore, the cell cycle time of satellite cells in growing pigs
was not established in this study, but may be an additional mechanism by which

the activity of satellite cells may be modulated.

48

Chapter 2

Tables and Figures

49

Table 2.1. Antibodies used to quantify porcine satellite cell proliferation and
differentiation.

50

 

02 .0 :0 5E0

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03:00.0”. 9.8000090 0:0 :o09000n at: :mflfli >0ona:<

 

 

51

Figure 2.1. Liveweight (kg) of growing pigs at 1, 7, 14, and 21 weeks of age.
Error bars represent standard error of the mean (SEM). When not shown error
bars are smaller than the marker.

52

 

 

 

 

100

90.
804

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Figure 2.2. Panel A: left semitendinosus muscle weight (g) and total DNA (mg)
of growing pigs at 1, 7, 14, and 21 weeks of age. Panel 8: left semitendinosus
muscle weight (g) and total protein (9) of growing pigs at 1, 7, 14 and 21 weeks
of age. Error bars represent standard error of the mean (SEM). When not
shown error bars are smaller than the marker.

54

3.5 <20 .58

 

 

 

 

 

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14

Age (weeks)

55

Figure 2.3. Panel A: right Iongissimus dorsi muscle weight (g) and total DNA
(mg) of growing pigs at 1, 7, 14, and 21 weeks of age. Panel B: right Iongissimus
dorsi muscle weight (g) and total protein (9) of growing pigs at 1, 7, 14, and 21
weeks of age. Error bars represent standard error of the mean (SEM). When
not shown error bars are smaller than the marker.

56

Longissimus Dorsi weight (g)

Longissimus Dorsi weight (g)

 

 

 

 

 

 

1800 1400
1600 - A +1.0 weight - 1200
1400 - - Q. Total DNA
. 1000
1200 - ‘3‘
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800 - - 600 g
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Age (weeks)
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Age (weeks)

57

21

Figure 2.4. Daily myonuclear accretion of the left semitendinosus muscle of
growing pigs from 1 to 7, 7 to 14, and 14 to 21 weeks of age. Number of nuclei
accumulated per day calculated based on 7.4 pg DNA/nucleus (Doumit et al.,
1993).

58

 

 

 

       
 

 

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Weeks of age

Figure 2.5. Protein to DNA ratio of the left semitendinosus and right Iongissimus
dorsi muscles of growing pigs at 1, 7, 14 and 21 weeks of age. Error bars
represent standard error of the mean (SEM). When not shown error bars are
smaller than the marker.

60

 

 

 

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61

Figure 2.6. Fluorescent micrographs of porcine satellite cells stained for the
presence of neural cell adhesion molecule (NCAM). Micrographs are presented
in color and at 400x magnification. Panel A: Detection of total nuclei
accomplished by counterstaining cells with 4’,6—diamidino—Z-phenylindole (DAPI).
Panel 8: Representative immunostaining for NCAM. Closed and open arrows
indicate positive and negative staining, respectively.

62

 

Figure 2.7. Fluorescent micrograph of porcine satellite cells stained for the
presence of proliferating cell nuclear antigen (PCNA). Micrographs are
presented in color and at 400x magnification. Panel A: Detection of total nuclei
accomplished by counterstaining cells with 4’,6-diamidino-2-phenylindole (DAPI).
Panel 8: Representative immunostaining for PCNA. Closed and open arrows
indicate positive and negative staining, respectively.

64

 

Figure 2.8. Fluorescent micrographs of porcine satellite cells stained for the
presence of myogenin. Micrographs are presented in color and at 400x
magnification. Panel A: Detection of total nuclei accomplished by counterstaining
cells with 4',6-diamidino-Z-phenylindole (DAPI). Panel B: Representative
immunostaining for myogenin. Closed and open arrows indicate positive and
negative staining, respectively.

66

 

Figure 2.9. Fluorescent micrographs depicting representative non-specific
immunostaining for NCAM, PCNA, and myogenin. Micrographs are presented in
color and at 400x magnification. Panels A, C, and E: Detection of total nuclei
accomplished by counterstaining cells with 4’,6-diamidino—Z-phenylindole (DAPI).
Panel 8: Representative non-specific immunostaining for NCAM. Panel D:
Representative non-specific immunostaining for PCNA. Panel F: Representative
non-specific immunostaining for myogenin.

68

 

69

Figure 2.10. Changes in the percentage of satellite cells (cells expressing
NCAM) isolated from the right semitendinosus muscle of growing pigs at 1, 7, 14,
21 weeks of age. Error bars represent standard error of the mean (SEM).
Different superscripts indicate means that differ (P <0. 05).

70

 

 

 

    
 
     

  

 

 

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pigs at 1, 7, 14, 21 weeks of age. Error bars represent standard error of the
mean (SEM). Different superscripts indicate means that differ (P <0. 05).

76

 

 

 

 

 

 

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Age

CHAPTER THREE

RECOMMENDATIONS FOR FUTURE RESEARCH

It appears that in the rapid phases of porcine skeletal muscle growth (1 to
21 weeks of age), satellite cells retain a high level of proliferation (greater than
75% PCNA+ satellite cells) whereas the proportion of satellite cells that are
capable of differentiation decline within this period. Furthermore, the percentage
of satellite cells to non-myogenic cells isolated from the ST muscle declined from
1 week of age and remained at constant levels thereafter. Thus, the generally
accepted theory that satellite cell activity declines as these cells enter a
quiescent state does not apply to growth of porcine ST muscle from 1 to 21
weeks of age. Future studies should characterize satellite cell proliferation and
differentiation at time-points beyond than 21 weeks of age in order to determine
the influence of satellite cell activity at the stages in which ST muscle growth
plateaus. Moreover, future studies should examine the changes in satellite cell
activity of the LD muscle from 14 to 21 weeks of age. The observed decrease in
the rate of growth of the LD at these time-points (Figures 2.3.) would serve as an
excellent experimental model to characterize satellite cell activity at a point in
time where a relatively slower rate of muscle growth is observed.

Since PCNA is expressed in multiple stages of the cell cycle (Morris and
Mathews, 1989), it is important to note that the current immunostaining system
used to quantify proliferating satellite cells does not provide information about cell

cycle time of PCNA positive nuclei. The possibility arises that changes in

78

proliferation due to age may occur from a lengthening cell cycle and not from
changes in the number of cells progressing through the cell cycle at any time.
Bischoff (1986b) observed that satellite cells have a cell cycle time of
approximately 12 hours when exposed to mitogens released from an extract of
crushed muscle in vitro. On the other hand, Schultz (1996) demonstrated that
80% of satellite cells from growing rat muscle proliferate with an estimated 32-
cell cycle time with the remaining 20% proliferating more slowly. Taken together,
these data indicate that cell cycle time may be another regulatory checkpoint for
satellite cell activity and that populations of satellite cells with different
proliferative potentials may exist in growing muscle. Future studies should
examine if changes in satellite cell cycle time occur from 1 to 21 weeks of age as
well as the effect of these changes on skeletal muscle hypertrophy.

The mechanisms responsible for the changes in satellite cell activity
observed in the preceding chapter are unclear. The IGF-l system may play a
major role in the regulation of satellite cell activity. Insulin-like growth factor-I is
unique because it is able to stimulate both proliferation and differentiation of
myoblasts (reviewed by Florini et al., 1996). Insulin-like growth factor-I is also
demonstrated to act synergistically with FGF-2 to stimulate both proliferation of
porcine satellite cell clones (Doumit et al., 1993) and differentiation of rat satellite
cells (Allen and Boxhom, 1989). Possible mechanisms by which IGF-l may elicit
a decline in differentiation are 1) levels of IGF-I protein in skeletal muscle decline
with age, 2) levels of circulating or locally produced lGFBPs increase with age, 3)

levels of IGFR1 on the surface of satellite cells decline with age, 4) the affinity of

79

IGFR1 for its ligands decline with age, and/or 5) other growth factors that inhibit
the myogenic actions of IGF-I increase with age.

Mitogenic growth factors, such as FGF and HGF, may also be responsible
for the high degree of proliferation observed in this study. It is unknown if levels
of these other growth factors decrease with age in porcine muscle and if the
bioactivity of such peptides changes with development. Furthermore, it is
unknown if these other growth factors interact with IGF-l to elicit the observed in
vivo levels of proliferation.

In order to elucidate the mechanisms governing skeletal muscle
hypertrophy, it will be necessary to further characterize the proliferating and
differentiating populations of satellite cells. Furthermore, it will be important to
gain insight into the hormonal control of satellite cell activity, especially control by
locally produced growth factors. Such information will be useful in determining
the role of satellite cell proliferation and differentiation in DNA accretion of
skeletal muscle, ultimately leading to the development of strategies to optimize

accretion of lean tissue in meat animal species.

80

 

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81

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