Effect of exercise on the mRNA expression of growth factors, metabolic genes and myosin heavy chain isoforms in skeletal muscles of the rat

Matsakas, Antonios

Titel: Effect of exercise on the mRNA expression of growth factors, metabolic genes and myosin heavy chain isoforms in skeletal muscles of the rat

Effect of exercise on the mRNA expression of growth factors, metabolic genes and myosin heavy chain isoforms in skeletal muscles of the rat

Zusammenfassung

Inhaltsangabe:Abstract:
Skeletal muscle is a complex and heterogeneous tissue capable of remarkable adaptation in response to various stimuli such as exercise training. 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 response to a cellular stimulus is dependent on the dynamics of mRNA turnover. Changes in muscle mRNA steady-state levels can be taken as an index of changes in gene expression, constituting currently one of the best described and understood molecular events that underlie muscle plasticity.
The discovery of new growth factors involved in the regulation of muscle development provides a better understanding of the molecular mechanisms involved in the adaptation of skeletal muscle to exercise training. Since it has been shown that changes in contractile function can be brought about by switching on one subset and repressing another subset of genes, it was hypothesized that the steady state level of multiple mRNAs (growth factors and metabolic genes) involved in regulatory functions in a muscle which is frequently recruited during exercise is different between endurance trained versus untrained rats. Recent scientific data indicate that myostatin constitutes a limiting factor in normal muscle development playing a crucial role in skeletal muscle hypertrophy. Nevertheless, there are only scarce data so far about the role of myostatin in the exercise-induced skeletal muscle adaptation. The transient changes in regulatory and structural gene expression provide the molecular basis of the adaptability of the skeletal muscle to exercise stimulus.
To get an insight into the molecular mechanisms of skeletal muscle adaptation, the main objective of this study was to examine the effect of both the short- and long- term effect of exercise (five day of swimming vs. chronic wheel running) on IGF-I and MSTN (positive and negative skeletal muscle regulators respectively) mRNA contents in male Wistar rats. Potential increase of positive and/or decrease of negative regulators of muscle growth lead to enhanced muscle-progenitor proliferation providing a new perspective in the mechanism of muscle adaptability. It was hypothesized that exercise training could be […]

Leseprobe

Inhaltsverzeichnis

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

Table of contents

ACKNOWLEDGEMENTS... I

TABLE OF CONTENTS... II

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

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

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

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

method (C

) 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

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

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

SUMMARY... 87

REFERENCES ... 90

APPENDIX ... 105

7.1

List of Figures ... 105

7.2

List of Tables... 108

7.3

List of Abbreviations... 109

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

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

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

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

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

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

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.

Genes

Adaptation

Metabolic genes

CPT-I

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.

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.

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.

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.

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).

1.4.2.2 Structure

myostatin

Fig. 7. Human and mouse myostatin consensus

(from http://bighorn.animal.uiuc.

edu/Myostatin_files/image006.gif, accessed on 11.03.04)

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

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

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).

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

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

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).

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

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.

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

Details

Seiten
Erscheinungsform: Originalausgabe
Jahr: 2004
ISBN (eBook): 9783832482930
ISBN (Paperback): 9783838682938
DOI: 10.3239/9783832482930
Dateigröße: 2 MB
Sprache: Englisch
Institution / Hochschule: Deutsche Sporthochschule Köln – Medizin- und Naturwissenschaften, Kreislaufforschung und Sportmedizin
Erscheinungsdatum: 2004 (September)
Note: 1,0
Schlagworte: sport muskelphysiologie polymerasekettenreaktion wachstumsfaktoren myostatin

Autor

Antonios Matsakas (Autor:in)

Effect of exercise on the mRNA expression of growth factors, metabolic genes and myosin heavy chain isoforms in skeletal muscles of the rat

Zusammenfassung

Leseprobe

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Details

Autor

Antonios Matsakas (Autor:in)