ID 8293
Matsakas, Antonios: Effect of exercise on the mRNA expression of growth factors, metabolic
genes and myosin heavy chain isoforms in skeletal muscles of the rat
Hamburg: Diplomica GmbH, 2004
Zugl.: Deutsche Sporthochschule Köln, Dissertation / Doktorarbeit, 2004
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__________________________
Antonios Matsakas
iii
Acknowledgements
This study was performed in the Department of Molecular and Cellular Sports Medicine, Institute of
Sports Medicine and Cardiovascular Research, German Sport University Cologne during years 2001-
2004.
This work could never have happened without the support and assistance of many people. First and
foremost, I thank my advisor in the practical part of the work in the lab, Dr. Patrick Diel, for his
invaluable advice and encouragement in the past four years we have worked together. Special thanks
belong also to the rest of my committee--Prof. Dr. Hans-Joachim Appell and Prof. Dr. Hans-Georg
Predel--for their attention and advice. They have given much of their time and expertise through the
entire doctoral process. I would like to thank Prof. Dr. Vassilis Mougios from Aristotle University of
Thessaloniki, for the important support and his input in the blood hormone assays.
The financial support of the State Scholarships Foundation of Greece (IKY) is greatly acknowledged too.
Without it, it could have not been possible to start my doctoral studies in the German Sport University
of Cologne.
I would like to express my thanks to all colleagues of the department and friends for their constructive
and wholesome discussions and of course for the nice working atmosphere; factors with invaluable
importance for the successful completion of the present work. I acknowledge administrative and
technical staff members of the University, who have been kind enough to advice and help in their
respective roles. I am deeply grateful to my friends, who have sustained me through the trials and
tribulations of the doctoral process.
At last but not least, I would like to thank my parents for their unending mental and moral support
during all these years. My doctoral thesis is especially dedicated to my mother by referring to a verse from
my native language:
µ µ,
µ µ
µ . (. )
Antonios Matsakas
July 12th 2004, Cologne, Germany
i
Table of contents
ACKNOWLEDGEMENTS... I
TABLE OF CONTENTS... II
1
INTRODUCTION ... 1
1.1
Skeletal muscle adaptation to exercise: a historical perspective... 1
1.2
Gene expression in exercise ... 2
1.3
Useful tools to study gene expression... 4
1.4
Growth factors... 8
1.4.1
Insulin-like growth factor I... 8
1.4.2 Myostatin... 9
1.4.2.1 History of myostatin... 10
1.4.2.2 Structure of myostatin ... 12
1.4.2.3 Physiology of myostatin... 13
1.4.2.4 Myostatin and exercise... 17
1.5
Metabolic genes... 18
1.5.1 Hexokinase
II
mRNA... 18
1.5.2
Glucose transporter 4 mRNA ... 19
1.5.3 Hydroxyacyl-CoA-dehydrogenase
mRNA ... 22
1.6
Myosin heavy chain isoforms ... 22
1.7
Purpose of the study... 24
2
MATERIALS AND METHODS... 26
2.1
Animals... 26
2.2
A methodological pilot study ... 26
2.3
Training protocols ... 26
2.3.1 Short-term
swimming... 26
ii
2.3.2
Long-term wheel running... 29
2.4
Muscle dissection ... 29
2.5
mRNA analysis ... 32
2.5.1 TRIzol
Reagent total RNA Isolation Method ... 32
2.5.2
First-strand cDNA synthesis from total RNA ... 35
2.5.3 Semiquantitative
reverse
transcription polymerase chain reaction ... 37
2.5.3.1 Principle of the PCR... 38
2.5.3.2 Effect of the PCR components on the reaction ... 41
2.5.4
Real time RT-PCR... 41
2.5.4.1 The choice of suitable reference gene ... 44
2.5.4.2 The comparative C
T
method (C
T
) for relative quantitation of gene expression45
2.5.4.3 Primer design... 47
2.6
Blood hormone concentrations... 48
2.6.1
IGF-I assay in blood... 48
2.6.2
Testosterone assay in blood... 51
2.7
Statistical analysis... 52
3
RESULTS... 53
3.1
Methodological pilot study ... 53
3.1.1
Establishment of the detection system ... 53
3.1.2
Effect of the point time of muscle excision on RNA quality ... 53
3.1.3
Testing the primers... 55
3.1.4
Effect of exercise on the mRNA contents of reference genes... 55
3.1.5
Use of real-time RT-PCR ... 57
3.2
Short-term swimming: Regulatory, metabolic and structural genes ... 58
3.3
Long-term wheel running ... 65
3.3.1
Body weight and running activity ... 66
3.3.2
Regulatory, metabolic and structural genes mRNA levels and serum hormones . 67
4
DISCUSSION... 72
iii
4.1
Reference genes served as reliable controls for normalization ... 72
4.2
Modes of exercise: Short-term swim training and long-term wheel running 74
4.3
Effect of exercise on mRNA levels of metabolic genes ... 74
4.4
Effect of exercise on MHC isoform mRNA profile ... 77
4.5
The role of myostatin and IGF-I in the adaptation of skeletal muscle to
endurance exercise... 77
4.6
Muscle specific expression ... 80
4.7
Current approaches and future directions: Pharmacological strategies for the
inhibition of myostatin signaling and the risk of doping ... 81
5
SUMMARY... 87
6
REFERENCES ... 90
7
APPENDIX ... 105
7.1
List of Figures ... 105
7.2
List of Tables... 108
7.3
List of Abbreviations... 109
iv
1 Introduction
For centuries, scientists have studied how the human body works. Advances in our
understanding of training adaptations have come in waves caused by the introduction of
new experimental approaches. Exercise researchers in the past century have been
motivated by the desire to understand how the biological limits of the human body can be
extended and much progress in this area has been accomplished studying the skeletal
muscle training adaptations. Although research of the latter half of the 20th century has
revealed that exercise may effectively prevent some of the most common chronic diseases,
most of the mechanisms underlying the adaptation of skeletal muscle to exercise still
remain to be clarified.
1.1 Skeletal muscle adaptation to exercise: a historical perspective
The principle that muscle mass adapts during progressive strength training had been well
established by the time of the Greek and Roman dynasties, but until the 1500s any truly
significant contributions had not been done to understand both the structure and function of
the human body (Wilmore and Costill 1999). In 1777, Lavoisier demonstrated that oxygen
is taken in by the body and carbon dioxide is given off during respiration. In 1842, it was
shown that protein, carbohydrate, and fat were oxidized by the body, but the first definitive
association of carbohydrate metabolism with muscle contraction was not made until 1867.
The caloric values of fat, protein, and carbohydrate were provided in 1884 and calorimetry
studies were common by the end of the 19th century (Hamilton and Booth 2000).
At the outset of the 20th century, August Krogh developed instruments such as the tilting
spirometer, the electromagnetic bicycle ergometer and an apparatus for gas analysis. In
1920, he won the Nobel Prize in Medicine and Physiology for his discovery on the
regulation of the motor mechanism of capillaries, and by extending his work to exercise he
found that the average diameter of open microvessels was wider in working than in resting
muscles. Shortly thereafter, in 1922, Archibald Hill was awarded the Nobel Prize for his
findings relating to the production of heat in muscle (Hamilton and Booth 2000). At that
time, biochemistry was in its infancy, though it was rapidly gaining recognition through
the research efforts of such Nobel laureates as Albert Szent Gorgyi, Otto Meyerhof, and
Hans Krebs, who were all actively studying how living cells generate energy. However,
until the late 1960s, most exercise physiology studies focused on the whole body's
response to exercise. The majority of investigations involved measurements of such
1
variables as oxygen uptake, heart rate, body temperature and sweat rate. Cellular responses
to exercise received little attention (Wilmore and Costill1999).
Many advancements in the field of exercise physiology must be credited to improvements
in technology. The reintroduction of the muscle needle biopsy, in 1966, for exercise
research by Bergström and Hultman had a major impact on the following years of research
(Bergström and Hultman 1966), by allowing direct measurements of muscle chemistry.
Muscle biopsies and histochemistry allowed for the study of the metabolic characteristics
of fiber type, muscle fiber type composition, and myosin isoforms' turnover in response to
endurance and strength training.
Within the last 30 years,
exercise-related research has rapidly transitioned from an organ
to
a subcellular/molecular focus. With
the introduction of an improved muscle-biopsy
technique and the
ability to invasively study small laboratory animals (e.g., rodents) during
acute
and chronic exercise, the focus rapidly shifted to analyses at
the cellular and
subcellular level. This advancement was facilitated
via the use of a variety of evolving
biochemical techniques, radioisotope
and imaging technologies, which collectively enabled
studies of
organelle and cellular functions. These include gene-cloning and -sequencing
technology,
molecular probing via antibodies and oligonucleotides, the use
of polymerase
chain reaction (PCR) technology, as well as the development of transgenic and
gene
knockout animal models (Baldwin 2000).
1.2 Gene expression in exercise
Skeletal muscle is a complex and heterogeneous tissue capable of remarkable adaptation in
response to various stimuli such as exercise training. Alterations in the loading state
chronically imposed on skeletal muscles are known to result in remodeling processes,
which can be measured as changes in muscle mass and can be also detected at the cellular
and molecular levels in the myofibers of affected muscles.
The role of gene transcription,
as an initial target to control protein synthesis, has been proven as a major challenge in
molecular muscle research (Flück and Hoppeler 2003, Reecey et al. 2003, Williams and
Neufer 1996). Molecular
biology approaches have made a large contribution to our current
understanding of how mechanical loading can alter gene expression
and muscle protein
synthesis rates in skeletal
muscle. The steady-state level of a given mRNA is a function of
both its rate of synthesis and its rate of degradation, implying that the rate of change in
2
response to a cellular stimulus is dependent on the dynamics of mRNA turnover (Williams
and Neufer 1996), (Fig. 1). Changes in muscle mRNA steady-state levels can be taken as
an index of changes in gene expression and currently appear to be the best described and
understood molecular events that underlie muscle plasticity (Flück and Hoppeler 2003).
Fig. 1. Potential mRNA changes. Potential changes in mRNA levels in response to a
stimulus. A) The mRNA level may rise and remain elevated. B) The mRNA level may
rise then return back to baseline levels (i.e., immediate response gene). C) The mRNA
level may rise in a delayed fashion and then return to a baseline level of expression.
D) The mRNA level may rise in a delayed manner and remain elevated. E)
Alternatively, the mRNA level may not change. The mirror image of each of these
response lines is also possible. (adopted from Reecey et al. 2003).
To understand how genes and exercise can interact to modify a phenotypic trait or health
outcome, it is necessary to consider
multiple levels of interaction. Bray reported that there
are many
complex ways in which genes and exercise, both together and separately,
can
influence the health status of an individual (Bray 2000). According to this model exercise
may both produce direct and immediate effects on health status,
without necessarily
altering gene expression or function or may affect health status indirectly by altering the
expression or action of one or more genes that influence
intermediate phenotypes (e.g.,
cholesterol level) that ultimately
produce disease outcomes. Biological interaction, in
which multiple genetic and
environmental factors are interconnected in complicated ways,
is inherent in complex chronic diseases, such as cardiovascular
disease. Because of the
3
complex nature of many diseases and intermediate
phenotypes, and of health status in
general, the model in figure 2 necessarily includes other genes and other environments, in
addition to exercise, that may or may not interact with one another
in determining overall
health.
Fig. 2. Model of gene-exercise interaction illustrates the complex interaction among
exercise, genes, and other environmental factors in overall determination of health
status. (used by permission, from Bray MS. Genomics, genes, and environmental
interaction: the role of exercise.
J Appl Physiol 88: 788-792, 2000).
1.3 Useful tools to study gene expression
Since v
ertebrate skeletal myofibers are heterogeneous with respect to their metabolic,
contractile and morphological properties, the specific reaction of a trained skeletal muscle
can by studied through the analysis of local gene expression. A whole-genome gene
expression analysis of skeletal muscle may be carried out, in order to better understand
skeletal myofiber diversity and plasticity at the transcriptional level (Wu et al. 2003).
Microarrays represent a novel genetic platform which is being widely exploited to bridge
the gap between gene sequence and function. Microarray technology possesses the ability
to simultaneously interrogate the whole genome on a single chip (Fig. 3). The so-called,
microarrays are simply ordered sets of DNA molecules of known sequence offering the
possibility to quickly analyze expression levels of known genes and providing a better
picture of the interactions among thousands of genes simultaneously. Microarray
4
technology has found so far broad use in the areas of disease diagnosis, pharmacogenomics
and toxicogenomics, and many opportunities continue to be created (Rouse and Hardiman
2003).
Fig. 3. A representative microarray chip. It can record which of the genes are active in a
cell, offering unprecedented opportunities as a future diagnostic tool. The intensity
and color of each spot encode information on a specific gene from the tested
sample. (
from http://www.gene-chips.com/sample1.html, accessed on 12.05.2004
)
This emerging technology has the potential to find important applications in sport science
allowing gene expression profiling. It is to expect that the use of microarrays in exercise
physiology will provide new insights into the complex molecular mechanisms of the
exercise-induced stress response, adaptation to training and modulation of molecular
function of the cell. Gene expression profiling and genetic screening will probably help to
characterize and predict the individually variable response to and efficiency of training
(Fehrenbach et al. 2003).
Genetic gene expression profiles based on individual
characteristics can be compiled and they may be correlated with the response to the
5
training program. This provides a useful tool to identify talents or athletes with probable
genetical predisposal to sport. It means that there is the possibility to define the
physiological reactions of a tissue to exercise stimuli and subsequently to check the
suitability of a training program.
Apart from microarrays, which provide the best technological tool to screen the whole
genome, quantitative RT-PCR (reverse transcription polymerase chain reaction) possesses
the possibility to both identify and quantify slight alterations in the expression of a short
number of genes (Freeman et al. 1999). The technique relies on the fluorescence of a
reporter molecule that increases as product accumulates with each cycle of amplification
and allows the user to quantify starting amounts of nucleic acid template by analyzing the
amount of DNA produced during each cycle of PCR.
In this era of molecular biology there is a tendency to assume that the development and
growth of tissues is pre-programmed in the genome. However, it is well established that
many cell types respond to mechanical as well as chemical signals. Skeletal muscle
constitutes the best example of a tissue that can adapt by qualitatively and quantitatively
changing gene expression in response to functional demands. Besides, it has been
appreciated for many years that there is local as well as systemic control of tissue growth
and that autocrine and paracrine mechanisms play a major role in determine muscle growth
as well as muscle adaptation in transcriptional level (Goldspink and Yang 2001). In
particular, changes in expression of myofibrillar and metabolic proteins have often been
demonstrated to be involved in skeletal muscle plasticity (Baar et al. 2002, Flück and
Hoppeler 2003, Goldspink and Yang 2001). Figure 4 illustrates various regulatory,
metabolic and structural genes, which play a significant role in muscle cell homeostasis.
6
Genes
Adaptation
Metabolic genes
CPT-I
CS
FABP
GLUT4
GYS
HAD
HK II
HO-1
LCAD
LPL
PDK-4
PFK
UCP3
Structural genes
Actin isoforms
MHC isoforms
Exercise
Regulatory genes
cFos
FGF
IGF-I
IGFBPs (1-6)
MGF
MSTN
Myf-5
MyoD
Myogenin
TGF-b
VEGF
Fig. 4. Genes which regulate muscle cell function and adaptation to exercise. The genes
set in bold are of special interest in this study.
Abbreviations: CPT-I: carnitine palmitoyl-transferase I, CS: citrate synthase, FABP:
fatty acid-binding protein, FGF: fibroblast growth factor, GLUT4: glucose transporter 4,
GYS: glycogen synthase, HAD: hydroxyacyl-CoA dehydrogenase, HK II: hexokinase
II, HO-1: heme oxygenase-1, IGF-I: insulin-like growth factor I, IGFBPs: insulin-like
growth factor binding proteins (isoforms 1-6), LCAD: long-chain acyl-CoA
dehydrogenase, LPL: lipoprotein lipase, MGF: mechanogrowth factor, MHC: myosin
heavy chain, MSTN: myostatin, PDK-4: pyruvate dehydrogenase kinase-4, PFK:
phosphofructokinase, TGF-b: transforming growth factor b, UCP3: uncoupling protein
3, VEGF: vascular endothelial growth factor.
7
1.4 Growth
factors
The fact that muscle cells may express locally substances like IGF-I and fibroblast growth
factors (FGFs) in response to physical activity, highlights the role of growth factors in
skeletal muscle growth (Goldspink et al. 2002). Growth factors are proteins that bind to
receptors on the cell surface, with the primary result of affecting cellular proliferation
and/or differentiation. Many growth factors are quite versatile, stimulating cellular division
in numerous different cell types, while others are specific to a particular cell-type. Their
cellular responses are primarily mediated by binding to specific receptors (King and Feener
1998).
1.4.1 Insulin-like growth factor I
The discovery of new growth factors involved in the regulation of muscle development
provides a better understanding of the molecular mechanisms responsible for the
adaptation of skeletal muscle to exercise training. One of these growth factors is IGF-I,
which influences cell proliferation and differentiation as well as stimulates protein
synthesis in many tissues (Rosen and Pollak 1999). IGF-I is released from the liver and
other tissues, including skeletal muscle, and is an endocrine growth factor which possesses
both autocrine and paracrine action on a large number of tissues (Le Roith 1997, Willis et
al. 1997). It has been recently demonstrated that IGF-I activates satellite cells and induces
hypertrophy of skeletal muscle (Adams 1998). It has become increasingly clear that the
main function of IGF-I is to regulate cellular growth and metabolism (Zapf et al. 1986).
Endurance training, apart from the well-described effects on the oxidative metabolic
machinery, can also elicit muscle hypertrophy (Gulve et al. 1993), and recent
investigations suggest that IGF-I might mediate this process (Adams 1998). Table I
summarizes the effects of exercise training on IGF-I mRNA. It seems that there is no
consensus about the effect of exercise on IGF-I mRNA, since significantly increased (e.g.
Bamman et al. 2001, Scheinowitz et al. 2003), decreased (e.g. Psilander et al. 2003) or
unaffected (e.g. Eliakim et al. 2001, Psilander et al. 2003, Zanconato et al. 1994) levels are
reported in the literature.
8
Table I. Effect of exercise on IGF-I mRNA
Reference
Subjects-exercise
IGF-I mRNA alterations
Scheinowitz
et al. 2003
Two and six weeks of
swimming in rats
IGF-I mRNA significantly increased in
myocardium.
Bamman
et al. 2001
A single bout of
mechanical loading in
humans
IGF-I mRNA significantly increased in
vastus lateralis muscle.
Eliakim
et al. 1997
Five days of treadmill
training in rats
IGF-I mRNA remained unaffected in
hindlimb skeletal muscle.
Psilander
et al. 2003
A single bout of heavy-
resistance training in
humans
IGF-I mRNA (isoform a) significantly
decreased but (isoform b and c) remained
unaffected in vastus lateralis muscle.
Zanconato
et al. 1994
A 4-wk treadmill training
program in rats
IGF-I mRNA remained unaffected in
hindlimb skeletal muscle.
1.4.2 Myostatin
A lot of attention has been recently focused on a new molecular parameter called myostatin
(MSTN). Myostatin, also known as Growth and Differentiation Factor 8 (GDF8), is a
member of the transforming growth factor-
(TGF-) superfamily of multifunctional
polypeptide growth factors that are involved in the regulation of cellular growth and
differentiation (Fig. 5) (Nicholas et al. 2002). Myostatin was identified in 1997 in the
mouse (McPherron et al. 1997a) and has been characterized as an important and potent
negative regulator of skeletal muscle growth (Ma et al. 2003, Hill et al. 2003) and inhibitor
of myoblast proliferation. For these reasons, this novel gene will be described in more
detail.
9
Fig. 5. TGF- superfamily
(from http://www.nature.com/nrm/journal/v1/n3/TGF_system/#i2, accessed
on 12.03.04)
1.4.2.1 History of myostatin
The phenotype of so-called "double-muscled" animals has drown the attention of
researchers for about two-hundred years. Examples of such animals are the Belgian Blue
and Asturiana de los Valles cattles, which are characterised by an enormous hypertrophy of
the skeletal muscle mass (Fig. 6). The Belgian Blue, for example, comprises about 45% of
the total cattle population in Belgium and is of enormous economical significance (Arnold
et al. 2001, Fiems et al. 1995). The molecular and genetical basis of the phenomenon of
"double-muscled" animals remained completely mysterious for a long time. Great progress
has been made during the past years through the development of transgenic technology.
Researchers were able to create so-called `'knockout"-mouse models, which enable them
to efficiently explore the biochemical pathways and mechanisms of gene action.
10
Fig. 6. Double-muscled breed of cattle lacking a functional myostatin gene.
(from
http://www.champion-nutrition.com/champion/products/myostatin/research.php, accessed on 10.12.
2003)
Using such techniques McPherron et al. identified the growth factor myostatin (1997a).
While looking for possible relatives of the well-known TGF- superfamily, they
discovered a novel gene with high homology to previously investigated members of the
family, being highly conserved among different species (Fig. 7). Subsequent targeted
mutation of the gene in mice resulted in an animal that showed a dramatic increase in size
and muscle mass (Fig. 8) (McPherron et al. 1997b). Accordingly, blocking of the MSTN
signalling transduction pathway by specific inhibitors and genetic manipulations has been
demonstrated to result in a dramatic and widespread increase in skeletal muscle mass due
to both hyperplasia and hypertrophy (McPherron et al. 1997b, Huet et al. 2001, Lee et al.
2001). Myostatin has been mapped to the same locus as that for muscle hypertrophy in
cattle (Smith et al. 1997). This locus is localised on the bovine chromosome 2 and via the
first successful bovine positional cloning experiment was shown that deletions in
myostatin gene were responsible for the double-muscled phenotype in cattle (Charlier et al.
1995, Grobet et al. 1997). Moreover, disrupted MSTN gene expression as a consequence
of naturally occurring mutations in cattle is associated with a hypermuscular phenotype
(Ma et al. 2003, Thomas et al. 2000).
11
1.4.2.2 Structure
of
myostatin
Fig. 7. Human and mouse myostatin consensus
(from http://bighorn.animal.uiuc.
edu/Myostatin_files/image006.gif, accessed on 11.03.04)
12
Myostatin belongs to the large TGF-
superfamily of signalling cytokines.
Members of the TGF- superfamily of
signalling cytokines have characteristic
sequence and structural patterns that dictate
their function. While some members of the
family regulate the cell growth by directing
the differentiation of neural tissue or by
inducing growth of mesenchymal cells,
other members may act as potent inhibitors
of cell proliferation, or modulate immune
responses, or promote erythropoesis (Piek et
al. 1999).
Fig. 8. Knockout myostatin mouse
(from
http://www.ast-ss.com/articles/article.asp?
AID =85, accessed on 10.12.2003)
Myostatin is in the carboxy-terminal region dissimilar enough, to defy classification into
any of the major TGF- subfamilies, such as inhibins, TGF-, and bone morphogenic
proteins, although it shares several characteristics in common with other members of the
superfamily (Arnold et al. 2001). Based upon recent findings, myostatin is synthesised as a
375-amino acid precursor protein that is proteolytically processed at an internal dibasic site
to give rise to a 26 kDa mature homodimeric protein. Proteolysis of myostatin precursor
results in an amino-terminal propeptide, and a 12.5 kDa carboxy-terminal fragment that is
the mature myostatin ligand (McPherron et al. 1997a, Rios et al. 2004, Thomas et al.
2000). It is important to note that on a genetic level the myostatin gene is extremely well
conserved throughout evolution. The inactive propeptide region is 90% identical, while the
mature protein is 100% identical among human, mouse, rat, pig, chicken, turkey, murine
and dog (Bogdanovich et al. 2004, McPherron et al. 1997b).
Myostatin is specifically expressed in the myotome layer of developing somites within the
myotome compartment during embryogenesis, and later in all skeletal muscles (Lee et al.
1999, McPherron et al. 1997b). After translation, the large TGF- superfamily precursor
molecules are delivered to the Golgi apparatus, where they are proteolytically cleaved by
endoproteases into an amino-terminal precursor remnant and a mature TGF-. Within the
Golgi apparatus, the amino-terminal precursor remnant interacts with latent TGF- binding
proteins to form large, latent complexes. These complexes are transported to the cell
surface, where they are activated by proteolytic cleavage of the amino-terminal precursor
remnant, which enable TGF- to bind to its receptors (Piek et al. 1999).
Accordingly, myostatin, like TGF-, exists as a large, latent complex with other proteins,
including its propeptide (Lee et al. 2001). Recent data obtained in vitro suggest that the
propeptide blocks myostatin binding to activin type IIB receptors, and in vivo increased
expression of the propeptide in transgenic mice results in increased muscle mass, due to
both increased muscle fiber number and size, indicating that the propeptide acts as a
myostatin inhibitor (Lee et al. 2001).
1.4.2.3 Physiology
of
myostatin
Myostatin has various physiological functions and constitutes a limiting factor in normal
muscle development. Given its pattern of expression: highly expressed in embryonic and
fetal stages of life and expressed to a lesser degree in adult muscle tissue, it can be
13
primarily viewed as a growth regulator in early development (McPherron et al. 1997a). In
spite of this variation of expression during phases of development, myostatin is expressed
in postnatal muscle tissue, and has been shown to affect adult tissue as well (Thomas et al.
2000). Myostatin is secreted by muscle cells and acting upon those cells to inhibit growth
(McPherron et al.1997a, McPherron et al. 1997b, Gonzalez-Cadavid et al.1998). The
inhibitory effects of myostatin have been shown to be reversible in vitro (Thomas et al.
2000), which tends to support the notion of myostatin as a reporter and regulator whose
effects can be modulated with changes in muscle tissue size and cell number.
Myostatin specifically affects muscle cells by possessing the ability to inhibit myoblast
proliferation through cell cycle control functions. Recombinant myostatin has been shown
to reversely inhibit C2C12 murine myoblast proliferation by arresting cells in the G1 and
G2/M stages of the cell cycle. In other words, myostatin is believed to control the G1 to S
and G2 to M transitions of the cell cycle through modulation of cyclin-dependent kinase 2
protein levels and p21cip1, a cyclin-dependent kinase inhibitor (Thomas et al. 2000).
Over-
expression of myostatin is believed to induce cell cycle arrest at the G1 stage followed by
termination of proliferation (Fig. 9).
Thomas et al. (2000) proposed a model for the role of myostatin in muscle growth, as
illustrated in the figure 9. During embryonic myogenesis, Myr-5 and MyoD genes specify
cells to adopt the myoblast fate. Myoblasts then migrate and proliferate. In response to
functional myostatin signaling, p21 is upregulated, inhibiting cyclin-E·dependent kinase
(
Cdk2) activity, which causes retinoblastoma inactivation and G1 arrest. Thus, myoblast
number and, hence fiber number, following differentiation, is limited (Fig. 10A). In the
absence of functional myostatin, the signal for p21 upregulation is lost and the Rb remains
in a hyperphosphorylated form, resulting in increased myoblast proliferation, which leads
to increased fiber number (Fig. 10B).
In support of this contention, overexpression of myostatin in transfected C2C12 cells
reversely inhibited the myogenic process by downregulating mRNA levels of the muscle
regulatory factors myoD and myogenin, as well as the activity of their downstream target
creatine kinase, implying that myostatin negatively regulates muscle mass not only by
decreasing the proliferation rate of myoblasts but also by inhibiting its terminal
differentiation (Rios et al. 2002).
14
Fig. 9. Proposed myostatin mechanism (adopted from Kelvin et al. 1989)
Note:
Eucariotic cells execute their reproduction in a cyclic process. A standard cell cycle is comprised of
four phases. G1 (Gap 1) phase includes the period between M (mitosis) and S (synthesis) phase, in the S
phase takes place the replication of nuclear DNA and thus the doubling of genetic information, G2 (Gap 2)
phase lies between S and M phase, and in M (mitosis) phase, division of the chromosomes between the
daughter cells is prepared and carried out (Krauss 2001).
It is important to note that overexpression of myostatin contributes to the pathophysiology
of muscle atrophy as judged from various physiological states such as, muscle wasting in
individuals infected with HIV (Gonzalez-Cadavid et al. 1998), glucocorticoid-induced
muscle atrophy (Ma et al. 2003), human disuse muscle atrophy (Reardon et al. 2001),
microgravity environment exposure (Lalani et al. 2000). Moreover, myostatin expression
patterns exhibit an upregulation in cardiomyocytes after heart damage (Sharma et al.
1999), hindlimb unloading-induced atrophy of fast twitch muscle in mice (Carlson et al.
1999), hindlimb unloading without significant muscle loss in rat (Wehling et al. 2000), in
15
necrotic fibres and connective tissue of muscle damaged by myotoxin (Kirk et al. 2000)
and downregulation in regenerating muscle (Sakuma et al. 2000).
Fig. 10. The role of myostatin in muscle growth. (adopted from Thomas et al. 2000)
Like all tissues of the body, the specialized cells assembling the developing muscle begin
as undifferentiated precursor cells or stem cells that commit to the mesoderm. Mesodermal
progenitor cells then undergo modulation by growth factors at the determination and
differentiation stages, which collectively assign the final identity of the cells (Kelvin et al.
1989). Members of the TGF- superfamily all act as positive and/or negative regulators of
points in the pathway that sculpt a myocyte from an undifferentiated stem cell, and they
operate by way of tissue specific cell-surface receptors (Kelvin et al. 1989). TGF-
regulates at the terminal differentiation level, blocking the differentiation of myoblasts
(Kelvin et al. 1989).
Myostatin specifically affects muscle cells. However, it is expressed in other tissues and
may carry out cell cycle control functions in these tissues, as well. For example, although
myostatin is expressed only at low levels in adipocytes (McPherron et al. 1997a), it has
been shown to inhibit the differentiation of preadipocytes into adipocytes, probably by
16
inhibition of transcription factors (Kim et al. 2001). Myostatin can thus be said to have a
direct effect on adipogenesis, in addition to its well-described indirect effects that result
from radically changing the ratio of muscle to adipose tissue.
1.4.2.4 Myostatin and exercise
Numerous studies have investigated the role of myostatin in muscle growth (Carlson et al.
1999, Wehling et al. 2000, Ma et al. 2003, Hill et al. 2003). The importance of myostatin
as a regulator of skeletal muscle mass has been established, first in myostatin null mutant
mice and subsequently in Belgian Blue and Peidmontese cattle. Recent evidence suggests
that myostatin may also have a function in the training-induced skeletal muscle adaptation,
illustrating once more its role in skeletal muscle growth (Carlson et al. 1999, Ivey et al.
2000, Kawada et al. 2001, Lalani et al. 2000, Roth et al. 2003, Walker et al. 2004, Wehling
et al. 2000). Previous studies have established that myostatin expression may increase in
fully differentiated, non-pathological skeletal muscle during unloading (Carlson et al.
1999, Wehling et al. 2000). Myostatin expression seems to be affected by muscle loading
after a period of atrophy. Animal studies revealed reduced myostatin expression from
elevated levels in response to reloading through treadmill training (Wehling et al. 2000) or
restoration of normal gravity and caging conditions after atrophy inducing conditions
(Kawada et al. 2001, Lalani et al. 2000).
Myostatin has, so far, been investigated only with regard to resistance training and
treadmill running (Wehling et al. 2000), in both animals and humans (Ivey et al. 2000;
Roth et al. 2003; Schulte and Yarasheski 2001, Walker et al. 2004) or during hindlimb
immobilization in mice (Carlson et al. 1999). Specifically, Roth et al. (2003) found a
significant myostatin mRNA reduction in the vastus lateralis of previously sedentary
healthy individuals, after 9 weeks of heavy resistant training. Moreover, recent evidence
suggests that high-intensity resistance training lead to decreased cirdulating levels of
MSTN in healthy men (Walker et al. 2004). Recapitulating the above mentioned findings it
is apparent that muscle loading is followed by decreased levels of local muscle myostatin
mRNA and protein, although we do not know at present whether the observed reduction of
myostatin mRNA contents are the cause or the result of the exercise stimulus. In addition,
MSTN has been associated with muscle fiber type, exhibiting higher amounts of mRNA
and protein in fast-twitch compared to slow-twitch muscle in mice, supporting its fiber
type specific function (Carlson et al. 1999).
17
1.5 Metabolic
genes
Endurance exercise training has been shown to elicit marked alterations in substrate
utilization during exercise, including increased expression of key metabolic
enzymes and
an overall increase in mitochondrial content (Holloszy 1967, Hood 2001). At the
biochemical level, these adaptations improve the
efficiency of substrate utilization during
exercise, enhance
the sensitivity of the respiratory control system, and increase
the overall
oxidative capacity of skeletal myofibers, leading to a greater utilization of lipids at the
expense of carbohydrate (Constable et al. 1987, Dudley et al. 1987, Hood 2001, Pilegaard
et al. 2000). This decrease in carbohydrate utilization is due to a decrease in muscle
glycogenolysis and a decrease in muscle glucose utilization (Richter et al. 1999). The
magnitude of change is directly related to the intensity,
duration, and frequency of exercise
during the training program
(Dudley et al. 1982) and may also be reflected in the
transcriptional level (Hildebrandt et al. 2003). From this point of view, training-induced
increases in the mRNA levels for a number of metabolic genes have been reported,
implying that pretranslational regulation takes place in response to exercise (Pilegaard et
al. 2000).
1.5.1 Hexokinase
II
mRNA
The four mammalian hexokinases, designated HK I through IV, are a family of closely
related enzymes that convert glucose to glucose 6-phosphate. This enzymatic step initiates
glucose metabolism and ensures the glucose concentration gradient that results in the
movement of glucose into cell through the facilitative glucose transporters. Although HK I
and HK II are both expressed in skeletal muscle and adipose tissue, HK II is the
predominant hexokinase isoform in rat skeletal muscle, which phosphorylates glucose,
providing substrate inside the cell for subsequent reactions (Osawa et al. 1996). O'Doherty
and coworkers (1996) reported increased expression of both HK II mRNA and protein
level in rat gastrocnemius in response to metabolic challenges imposed by a single bout of
treadmill exercise. Table II summarizes the effects of exercise training on HK II mRNA.
All studies listed in the table have reported increased HK II mRNA contents after short-
term muscle loading.
18
Table II. Effect of exercise on HK II mRNA
Reference
Subjects-exercise
HK II mRNA alterations
O'Doherty
et al. 1994
a single bout of treadmill
exercise in rats
HK II mRNA significantly increased in
soleus, gastrocnemius/plantaris, and white
vastus.
Hofmann &
Pette 1994
contractile activity in low-
frequency stimulated (10Hz, 24
h/d) rat fast-twitch muscle
HK II mRNA significantly increased after
12 h from the onset of stimulation.
O'Doherty
et al. 1996
a single bout of treadmill
exercise in rats
HK II mRNA significantly increased in
gastrocnemius muscle.
Koval
et al. 1998
a single bout of cycling exercise
at 90% of anaerobic threshold
heart rate in humans for 1 hour
HK II mRNA significantly increased 3 h
postexercise in vastus lateralis muscle.
Pilegaard
et al. 2000
60-90 min of exhaustive one-
legged knee extensor exercise
in humans
HK II mRNA significantly increased 4 h
postexercise in vastus lateralis muscle.
Pilegaard
et al. 2003
3 hours of two-legged knee
extensor exercise in humans
(after 4 weeks of one-legged
extensor exercise)
Significantly increased HK II mRNA in
the trained than the untrained leg prior to
exercise. HK II mRNA significantly
increased in the untrained (but not in the
trained leg) at 6 h of exercise recovery.
1.5.2 Glucose transporter 4 mRNA
During contractile activity, muscle uses glucose as source of energy, and muscle represents
the major disposal site for insulin-stimulated glucose metabolism during postprandial state
(Tomas et al. 2001). GLUT4 has been characterized as the most abundant and most
important glucose transporter in skeletal muscle (Richter et al. 1999). It has been proposed
that the stimulation of glucose uptake by exercise training may be due to increased glucose
transporter isotype 4 content (Kawanaka et al. 1997). A rapid increase in GLUT4
expression has been referred as an early adaptive response of muscle to exercise, appearing
to be mediated by pretranslational mechanisms (Ren et al. 1994). Exercise leads to
increased translocation and expression of GLUT4, which play an important role in the
19
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- , 2004, Effect of exercise on the mRNA expression of growth factors, metabolic genes and myosin heavy chain isoforms in skeletal muscles of the rat, Hamburg, Bedey Media GmbH, https://www.diplom.de/document/223489
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