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Wright Carpenter, Tamsin: New Approaches for the Treatment of Muscle Injuries
Hamburg: Diplomica GmbH, 2005
Zugl.: Deutsche Sporthochschule Köln, Dissertation / Doktorarbeit, 2005
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2
,,Hierdurch versichere ich: Ich habe diese Arbeit selbständig
und nur unter Benutzung der angegebenen Quellen und
technischen Hilfen angefertigt; sie hat noch keiner anderen
Stelle zur Prüfung vorgelegen. Wörtlich übernommene
Textstellen, auch Einzelsätze oder Teile davon, sind als Zitate
kenntlich gemacht worden"
,,Hierdurch erkläre ich, dass ich die ,,Leitlinien guter
wissenschaftlicher Praxis" der Deutschen Sporthochschule Köln
in der aktuellen Fassung eingehalten habe"
3
In no particular order, I would like to thank all of the following:
Prof. Dr. Hans Joachim Appell for making it possible for a Spanish/British student with
a Scottish degree to enter a co-supervised PhD research project between the DSHS in
Germany and the CNRS in France. Thank you for sharing your knowledge with me,
instructing me in the art of scientific writing and encouraging me throughout my thesis,
especially through the more difficult times. I won't forget the laughs we had... your
sense of humour definitely lightened up the working sessions!
Dr. Lluis M. Mir for allowing this spanglish nomad from Formentera to enter his lab.
For the unforgettable time I spent in the CNRS, all I learnt of scientific and human
value, your motivation and the enriching chats which started in Spanish, moved onto
French, ended in English and sometimes even in Catalan!.. somos una especie extraña!
Prof. Dr. Peter Wehling for giving me the chance and the idea to start this project in the
first place. I sincerely hope that the contents of this thesis will lead to an application in
orthopaedics in the future.
Dr. Paule Opolon and Elisabeth Connault for all their laborious help with the histology.
Paule, you were a great moral support and are a real inspiration for all women in
science.
Desire Challuau and Claire Bernat for teaching me how to work with the animals and for
helping me to lose my initial apprehension.
Marie-Anges Verjus for her interesting and inspiring company. I will never forget our
coffee chats and your motherly support to me throughout my time in the CNRS. In our
next lives we will be dancers...
Dr. Brian Mullan for his precious help in molecular biology. You saved me from making
time consuming mistakes. I am so grateful for everything I learned from you.
4
Franck André and Nassim Morsli for being very helpful in our lab team. Frank, I have
no doubt your great team spirit will one day make you a superb researcher.
Dr. Jean-Rémi Bertrand for his helpful tips with the molecular biology but also for all
the interesting time we spent in the rather cramped office dreaming up culinary delights,
travels and admiring the beauty of orchids!
Dr. Thierry Ragot for lending me his precious viral vector, for his willingness to help
and his genuine interest in my project.
Dr. Luis Lopez Lázaro for his technical tips and for his support in PharmaMar when the
idea of my PhD first came up. From medicine, through poetry to informatics- you are the
21
st
century man of many talents!
Dr. Rainer Drever for all the articles you ordered for me and for taking care of my
laptop. I am so happy that you are returning to be a doctor at last!
Peter F. Butenschön and Bernt Alt for helping me with the informatics and for giving me
a hand with many tedious technical issues. Peter, you saved me when my laptop decided
to give up on me!
Arnaud for very kindly helping with the printing of my thesis.
My parents for being there for me in every possible way. I feel very privileged to have
you both.
My brother for being a good friend to me despite the geographical distance which has
separated us over the past years. I hope we will always be there for each other Tavis. I
am proud to have a brother like you...
5
My grandfather for motivating me to take on this challenge and for passing on his
ambition to me. I have been spoiled with grandparents like you and grandma. She is not
by our side anymore but she lives on in our hearts...
Robert for convincing me to do a PhD in the first place. Thank you for supporting all my
indecisiveness and for believing in me. I would love to be a co-author with you one
day...
Philipp to whom I owe so very much for giving me the inner strength to go to Paris for
this project in the first place. You gave me the push to leave our nest so that I could
finish my PhD. Without you, I would never have made it...
Barney, my almost flat mate on 120 Rue Vieille du Temple, Paris. For being sweet and
spicy and for putting up with all that flamenco!
My dear friends, Lydia, Amelie, Sascha and Bozo for being there and encouraging me
through all the changes. You are like family to me.
Vincent for encouraging me on the final sprint towards the end of this marathon. I know
you will be as glad as I will be when I hand in this thesis. Ci aspeta tantissimo...
And now in one hour's time I'll be out there again.
I'll raise my eyes and look down that corridor four feet wide with ten lonely seconds
to justify my whole existence.
Colin Welland (1934), British screenwriter.
Harold Abrahams (Ben Cross), Chariots of Fire,
before running in the Olympics (1981).
6
Scanning the history of science, we see how keen has been the animus to investigate the
structure of contractile elements in the hope of discovering the secret mechanism of the
most important phenomenon of animal life - movement!
Studies on muscle tissue by anatomists and physiologists throughout the years number in
the hundreds. These studies have been conducted with diverse methods - each more
perfect than the last, as technology became enriched with instruments, more precise
methods of observation and measurement, and new contrivances capable of revealing
the most minute structural details - and conducted with diverse objectives, as general
notions on animal organization were modified. These studies represent many
generations' steadfast pursuit of the solution to a great problem in biology.
And still we are far from reaching the goal.
Emilio Veratti, 1902
7
TABLE OF CONTENTS
TABLE OF CONTENTS ... 7
ABBREVIATIONS ... 9
INTRODUCTION... 10
MUSCLE INJURIES... 11
O
VERALL IMPORTANCE
... 11
T
YPES OF INJURY
... 11
M
USCLE IMAGING
: D
IAGNOSIS AND FOLLOW
-
UP OF INJURIES
... 13
REGENERATION PROCESS ... 15
T
IME FRAME OF REGENERATION
... 15
S
ATELLITE CELLS IN REGENERATION
... 21
G
ROWTH FACTORS IN REGENERATION
... 28
C
YTOKINES IN REGENERATION AND INFLAMMATION
... 37
A
NIMAL MODELS OF INJURY
... 38
TREATMENTS... 41
C
ONVENTIONAL TREATMENTS
... 41
A
TTEMPTS WITH GROWTH FACTORS TO ACCELERATE REGENERATION
... 44
AIMS OF THE STUDY... 47
A
UTOLOGOUS
C
ONDITIONED
S
ERUM IN THE TREATMENT OF MUSCLE INJURIES
... 47
E
LECTROTRANSFER OF GENES INTO THE MUSCLE
... 49
S
TRUCTURE OF THE THESIS
... 50
MATERIALS AND METHODS ... 51
TREATEMENT OF MUSCLE INJURIES WITH ACS: ANIMAL STUDY ... 52
M
ICE
... 52
C
ONDITIONED SERUM
... 52
ELISA
TESTS
... 53
C
ONTUSION MODEL
... 54
H
ISTOLOGICAL PROCEDURES
:
SATELLITE CELLS
... 54
H
ISTOLOGICAL PROCEDURES
:
CENTRONUCLEATED MYOFIBERS
... 55
F
IBROSIS DETECTION
... 56
S
TATISTICAL
A
NALYSIS
... 56
TREATEMENT OF MUSCLE INJURIES WITH ACS: HUMAN PILOT STUDY ... 58
A
UTOLOGOUS CONDITIONED SERUM
(ACS) ... 58
ACS ELISA
TESTS
... 59
ACS S
AFETY TESTS ON SERUM
... 59
T
REATED PATIENT GROUPS
... 59
T
HERAPY REGIME AND EVALUATION OF RECOVERY
... 61
S
TATISTICAL
A
NALYSIS
... 62
ELECTROGENE TRANSFER INTO MOUSE MUSCLE ... 63
C
ONSTRUCTION AND PURIFICATION OF P
CMV
B
FGF (
OR P
VAX
B
FGF) ... 63
P
LASMID
DNA ... 68
8
M
ICE
... 69
C
ONTUSION MODEL
... 69
DNA
INJECTION
... 69
DNA
ELECTROTRANSFER
... 70
GFP
EXPRESSION
... 71
L
UCIFERASE ACTIVITY MEASUREMENT
... 71
Q
UANTIFICATION OF REGENERATION
(
SATELLITE CELLS
)... 72
Q
UANTIFICATION OF REGENERATION
(
CENTRONUCLEATED MYOFIBERS
)... 73
M
EASUREMENT OF
FGF-2
CONCENTRATION
... 74
S
TATISTICAL ANALYSIS
... 74
RESULTS ... 75
TREATMENT OF MUSCLE INJURIES WITH ACS: ANIMAL STUDY... 76
ELISA
RESULTS
... 76
H
ISTOLOGY RESULTS
... 77
Q
UANTITATIVE REGENERATION RESULTS
... 80
TREATMENT OF MUSCLE INJURIES WITH ACS: HUMAN PILOT STUDY... 87
A
UTOLOGOUS CONDITIONED SERUM GROUP
... 87
C
ONTROL GROUP
... 89
ELISA
TESTS
... 91
ELECTROGENE TRANSFER INTO MOUSE MUSCLE ... 92
E
LECTROTRANSFER LEVELS AFTER INJURY
... 92
FGF-2
CONCENTRATION
... 93
S
ATELLITE CELL ACTIVATION
... 95
R
EGENERATING MYOFIBERS
... 97
DISCUSSION AND PERSPECTIVES ... 100
TREATMENT OF MUSCLE INJURIES WITH ACS: ANIMAL STUDY... 101
TREATMENT OF MUSCLE INJURIES WITH ACS: PILOT STUDY ... 104
ELECTROGENE TRASFER INTO MOUSE MUSCLE ... 109
GENERAL CONCLUSIONS... 113
SUMMARY ... 116
BIBLIOGRAPHY ... 118
9
ABBREVIATIONS
ACS
Autologous conditioned serum
EGF
Epidermal growth factor
FGF-2
Basic fibroblast growth factor (also bFGF)
HARP
Heparin affin regulatory pepetide (pleiotropin)
HGF
Hepatocyte growth factor
HSPG
Heparin sulphate proteoglycan
IGF-1
Insulin growth factor-1
IL-1ß
Interleukin 1 beta
IL-1Ra
Interleukin 1 receptor antagonist
IL-4
Interleukin 4
IL-6
Interleukin 6
IL-7
Interleukin 7
IL-18
Interleukin 18
LIF
Leukemia inhibitory factor
mpc
Muscle precursor cell (myoblast)
MRF
Myogenic regulatory factor family
MRI
Magnetic resonance imaging
NGF
Nerve growth factor
PDGF
Platelet derived growth factor
TGF-ß1
Transforming growth factor beta 1
10
INTRODUCTION
11
MUSCLE INJURIES
Overall importance
Muscle injuries constitute up to 55% of all injuries sustained in sport events [126]. The
incidence and severity of these injuries is certainly greater in some sport modalities than
in others. Significant morbidity, such as early functional and structural deficits, reinjury,
atrophy, contracture, and pain, often occurs following muscle injuries, leading to loss of
training and competition time [12, 92, 167]. Healing of these injuries is a complex
phenomenon depending of multiple factors, which are both within and outside the
control of the clinician. On the whole, the best treatment regime has not yet been clearly
defined, and the recommended treatment regimens have varied widely, depending on the
severity of the injury [198].
Types of injury
Injuries to skeletal muscle during sport can occur by different mechanisms including
blunt trauma in the case of contusions
[18]
or stretch-induced injury in muscle strains
[69, 70]. Another type of injury are those induced by laceration to the muscle but these
are not particularly relevant in sport [74]. A further complication of muscle injuries is
the compartment syndrome, which generally occurs when tissues within an osteofascial
compartment are compromised by increased pressure within the compartment.
12
On the whole, more than 90% of muscle injuries are caused either by excessive strain or
contusion of the muscle, with strains being overall the most frequent [37, 70].
Contusions are caused by the impact with a blunt, non-penetrating object and are
common in all contact sports, particularly American football, hockey, and soccer [21].
The symptoms are often non-specific, and include soreness, pain with active and passive
motion, and a limited range of motion. The severity of the contusion is relative to the
extent of blood vessel breakage and the degree of muscle crush. The bleeding in muscle
contusions often occurs within the muscle substance [166]. Clinically, in the relatively
common thigh contusions, the severity is rated by the amount of passive knee motion
available 12 to 24 hours after injury. A mild injury is indicated by flexion of more than
90º, 45º to 90º of flexion indicates moderate injury whereas less than 45º is characteristic
of a severe injury [101, 167]. The West Point study found that the average disability
time, defined as inability to participate in full cadet activities, was 13 days for mild, 19
days for moderate and 21 days for severe quadriceps contusion injuries [101, 167].
Within the complications of contusion injuries the development of Myositis Ossificans
Traumatica (MOT), as a consequence of haematoma ossification, is one of the most
troubling. MOT is the formation of nonneoplastic bone or cartilage in soft tissue and it's
detection is critical because it significantly increases the number of days of disability
after contusion [31].
Muscle strain may be a consequence of eccentric exercise, when the muscle develops
tension during this type of lengthening contraction [204]. These injuries are especially
common in high-velocity situations in sports that require sprinting or jumping such as
basketball, American football, rugby or soccer [48, 69, 70]. The most susceptible
13
muscles are the biarticular muscles such as the rectus femoris, the hamstrings and the
gastrocnemius [21]. Interestingly, a high percentage of type II fibers or fast twitch fibers
has been attributed to make muscles susceptible to strains because of their ability to
contract fast and produce high forces [72]. Furthermore, most muscle strains occur at or
very near to the myotendinous junction (MTJ) of the superficial muscles working across
two joints [69]. In the worst case, muscle stretch-induced injuries can lead to muscle
ruptures or tears. Jarvinen et al [105] have classified muscle strains into three categories
according to their severity: Mild (first degree) strains where a few muscle fibers are torn,
with minor swelling and discomfort but no or only minimal loss of strength and
restriction of movements; Moderate (second degree) strains where there is a greater
damage of the muscle with detection of a bleeding in the MRI scan and a moderate but
not complete loss of strength; Severe (third degree) strain in which we find a tear
extending across the whole or most of the muscle belly, resulting in total loss of muscle
function. In contrast to muscle contusions, the haematoma can collect between the
muscle tissue and the surrounding fascial compartment as shown by ultrasound [61]. The
time to recovery after muscle strain depends naturally on the severity of the injury as
classified above. For example, after a hamstring strain, returning a patient to sport
without the risk of suffering from reinjury can take from two weeks to six months [46].
Muscle imaging: Diagnosis and follow-up of injuries
The diagnosis of sports-related injuries has traditionally been one of clinical judgement
and examination. However, nowadays, modalities including ultrasound (also known as
14
myosonography), and magnetic resonance imaging (MRI), have become more and more
important in both identifying and delineating the extent of injury.
The CT scan was in fact the first imaging technique available for the evaluation of
neuromuscular disorders
[145]
but its use in the context of muscle injury has been
limited. Since the posterior introduction of ultrasound, this technique has played an
increasingly important role in sports traumatology [153]. Initially, it can help determine
the extent of injury and further on it can help the physician to decide the safe moment at
which the athlete can return to training and competition. Still for the latter, this may
often be difficult to assess with ultrasound alone, and more often the physician will rely
on clinical examination and a lack of symptoms upon resumption of slight athletic
activity.
The ideal time for ultrasound examination is between 2 and 48 h after the trauma. This is
because before 2 h, the haematoma is still in formation and after 48 h, the haematoma
can be spread outside the muscle. Presence of haematoma is a key sign of muscle tear
[164] and in most cases it appears as a hypo- or anechoic circumscribed lesion.
On the whole, the easy availability, low cost and ease of examination makes ultrasound
a more widely used imaging technique than MRI (see below) for the follow-up of
lesions and detection of healing problems such as fibrosis or myositis ossificans.
There is no doubt, however, that the technique for investigation of muscle injury has
been largely improved with the advent of MRI which provides better soft tissue
resolution than CT and ultrasound
[34]
. MRI technique is used to precisely locate muscle
strain injuries and it allows the observation of oedema (perhaps indicating concomitant
15
inflammation) and possible haemorrhage with high quality images [181]. Because of the
expense of this technique, MR imaging is not used frequently in routine. However, there
is no doubt that MRI is a very good method for predicting recovery following muscle
strain injury [21, 156].
REGENERATION PROCESS
Time frame of regeneration
Despite the different injury mechanisms mentioned, the pathological events in the
muscle tissue repair are similar. In terms of the natural healing of muscle, animal
research has given us a good insight into this process, which involves a complex balance
between muscle repair, regeneration, and scar-tissue formation. The healing process is
characterised by three phases: (1) the destruction phase, characterised by haematoma
formation, myofiber necrosis or degeneration, inflammation and phagocytosis of the
necrotised tissue to facilitate the repair process; (2) the repair phase with regeneration
of the myofibers after activation of the muscle satellite cells, production of connective
tissue scar, and capillary ingrowth; (3) the remodelling phase, where the regenerated
myofibers mature, the scar tissue eventually disappears and the function of the repaired
muscle is restored [105]. The repair and remodelling often occur simultaneously
(described as healing phase ahead).
The time frame and the basic scheme of regeneration following injury to a fiber are
summarised in figures 1 and 2 ahead.
16
Figure 1. Summary of basic scheme of regeneration following injury to a fiber. In the first
week after injury there is an onset of degeneration of the damaged muscle fibers, followed
by an inflammatory response to remove the muscle debry and then by regeneration to
replace the damaged muscle fibers. The regeneration phase lasts at least 3 weeks post-
injury.
Acording to Lehto and Järvinen [126], the following factors will determine the rate of
healing after an injury takes place: impairment to the neuromuscular input, vascular
ingrowth, oxygen supply, rate and geography of myogenesis (see satellite cell section
below), collagen cross-linking, and most importantly the overall competition between
regenerating myoblastic cell infiltration and granulation and scar formation
17
Figure 2: Basic scheme of muscle regeneration after injury: (1) Trauma
affects myofiber; (2) Myofiber undergoes focal necrosis and autodigestion
but the endomysial tube and satellite cells remain; (3) Macrophages
penetrate the endomysial tube to remove necrotic debris while satellite
cells become activated; (4) The satellite cells proliferate and differenetiate
into myoblasts thereby withdrawing from the cell cycle; (5 and 6)
Myoblasts fuse to form multinucleated myotubes with subsequent fusion
to the surviving stumps; (7) Original myofiber is restored with its
complement of satellite cells (Bischoff R, 1994) [29]. More detailed
description in the text.
18
Destruction phase
Because of the rich vascularisation of skeletal muscle, haemorrhage from the torn
vessels is inevitable immediately post-trauma, filling the injured area with haematoma.
Also within minutes after injury, the myofibers undergo hypercontraction, forming a
dense irregular mass of myofibers which are visualised as "contraction clots" under the
microscope [143]. It is thought that this occurs because the mechanical trauma (Fig. 2 -
1-) destroys the integrity of the myofiber sarcolemma, leading to a rapid influx of
extracellular calcium and to myofiber necrosis and autodigestion (Fig. 2 -2-) [11, 40].
Once Ca
2+
has entered into the fiber it activates proteases called calpains which break
down a wide variety of myofibrilar and other cytoskeletal proteins found in muscle, such
as myosin, -actin , talin, and vinculin [62, 186]. Remarkably, in most types of injury the
endomysial tube (basal lamina and reticular lamina) surrounding the necrotised tissue of
the injured myofibers is preserved (Fig. 2 -2-) and spans the region between contraction
clots [28]. The endomysial tube contains remnants of myonuclei, mitochondria and
membrane fragments which disappear from cultured myofibers within 24 hours,
presumably by some kind of organellolysis [203].
Since the intrinsic degeneration can only remove a small portion of the necrotic tissue,
the phagocytic activity of the inflammatory response is essential for subsequent
regeneration [189]. Indeed, the remaining necrotic clots of myofilaments require mostly
the macrophage phagocytic activity for removal (Fig. 2 -3-). The timing and the rate of
the inflammatory response is proportional to the preservation of the vascular supply. A
large number of mononuclear cells are found within the endomysium and the damaged
muscle cells within the first 2 to 3 days after injury
[58]
. Initially, the neutrophils invade
19
the injury site and promote inflammation with the release of cytokines that can attract
and activate additional inflammatory cells [189]. Neutrophils can further damage the
injured muscle by releasing reactive species of oxygen. The response is followed by an
increase in macrophages (derived from blood monocytes) that complete the removal of
the necrotic debris. However, macrophages also have an essential role in stimulating
regeneration by secreting effector molecules (such as FGF, PDGF, IGF-1, IGF-II, HGF,
TGF , LIF, IL-4, IL-6, IL-10, IL-12, IL-18) that act on the satellite cells as mitogens,
induce chemotaxis, and promote revascularisation [161] (Fig. 6 ahead). It is not clear
what effector molecule provides the chemoattractive stimulus to inflammatory cells.
However, several growth factors and cytokines such as PDGF [52], IL-1 [155], TGF-ß
[160] and TNF- [116] secreted by fibroblasts and macrophages appear to have a role in
mediating the inflammatory response. Both degenerative and inflammatory responses
occur in the first week after injury.
Healing phase
During the healing phase there is a delicate balance between the regeneration of normal
muscle by migrating satellite cells and the formation of scar tissue by fibroblasts
(remodelling phase). The so called race between regenerating myoblastic cell infiltration
and connective tissue scar formation is said to be one of the most important factors in
the success of the healing process [126]. These processes are both supportive to but also
competitive with each other.
The endomysial tube, which completely encloses the myofiber and associated satellite
cells (see Fig. 3 ahead), plays an important role in regeneration, including (1) spatial
20
orientation of nascent myotubes, (2) promoting growth and differentiation, and (3)
furnishing essential signals for reinnervation. However, its most important function may
be to serve as a scaffold in which the satellite cells begin the myogenic process [97].
Thus the endomysial tube may facilitate regeneration by keeping most satellite cells
within a confined space and separate from other nonmuscle elements (except
macrophages) to promote their interaction.
At the origin of the regenerative process, we find the satellite cells. Their initial
activation takes place in the first 24 hours post-injury (Fig. 2 -3-). The cells proliferate
while attached to the inner surface of the basal lamina so that a "basophilic cuff" of
tissue is formed (Fig. 2 -4-). Already at day 2 to 3 post-injury, the satellite cells will
have differentiated into myoblasts and begin to withdraw from the cell cycle and fuse to
form multinucleated myotubes (Fig. 2 -5- and 2 -6-) [108]. The culmination of the
repair process of the myofiber occurs when the original myofiber and complement of
satellite cells are restored (Fig. 2 -7-).
At the origin of the remodelling process we find the mononuclear fibroblasts. The
presence of fibroblasts, mostly in the endomysial connective tissue, increases gradually
within the first week after injury [58]. Initially, the blood derived fibrin and fibronectin
form a matrix which acts as an anchorage site for the invading fibroblasts. The
fibroblasts synthesize both proteins and proteoglycans of the extracellular matrix
(ECM). Fibronectin and tenascin are expressed among the first ECM proteins followed
by type III collagen at day 2 to 3 post injury [108, 143]. The production of type I
collagen is activated later and remains elevated for several weeks. The tensile strength of
the scar increases simultaneously with the deposition of ECM. Indeed, the scar has the
21
essential physiological role of keeping the muscle stumps together, providing as well the
connective tissue to re-establish the firm attachment of the myofiber ends. However, the
problem in the regeneration process arises when the scar tissue formation is out of
balance with the myofiber repair process, thus impeding the muscle stumps to rejoin
efficiently and raising the probability of reinjury.
Satellite cells in regeneration
Myofibers are "post-mitotic" as they do not conserve the intrinsic capacity to proliferate
and generate new myofibers. Thus, in the mature muscular tissue, only the
mononucleated satellite cells are capable of proliferating and differentiating into muscle
fibers. This process requires the activation of the satellite cells and is vital for muscle
growth and regeneration.
Definition
Originally described by Mauro in 1961
[131]
, satellite cells are defined as
mononucleated cells lacking myofibrils and situated beneath the basal lamina of skeletal
muscle fibers (i.e. sublaminal or "under" the endomysial tube, see Fig. 3A)
[27, 172]
.
These myogenic precursor cells, react in response to muscle injury and proliferate,
giving rise to myoblasts which fuse to myotubes, and finally mature into muscle fibers, a
process known as myogenesis [81] (see Fig. 5 ahead).
22
23
Embryonic myogenesis is characterised by the differentiation of mesodermic somitic
cells into myoblasts (Fig. 3 -C-), which will then fuse to form polynucleated myotubes
(Fig. 3 -D-), the association of which gives rise to new muscle fibers (Fig. 3 -E-)[10]. In
the early phase of embryonic myogenesis (Fig. 4), Pax-3 expression in progenitor cells
contributes in amongst other factors to myogenic cell expansion and is a key regulator of
somatic myogenesis [185]. After Myf 5 and/or Myo D induction, mesodermal somatic
cells are committed to the myogenic lineage (myoblasts). Later, upregulation of the
secondary muscle regulatory factors (MRFs), myogenin and MRF 4, induces terminal
differentiation of myoblasts into myocytes. Finally, myocyte fusion gives rise to
multinucleated myofibers. During the later phase of embryonic myogenesis, a distinct
population of myoblasts, derived from satellite cells, fuses to existing myofibers
enabling myofiber growth. Some satellite cells remain closely associated with myofibers
in a quiescent undifferentitated state and these are the ones which are activated after
injury. Pax 7 expression is essential for the specification/expansion of the satellite cell
population. This is the gene responsible for the specification of progenitor cells to the
satellite cell lineage [175].
24
Figure 4. Signaling factors and cellular events involved in embryonic myogenesis
(modified from Chargé et al., 2004) [45]. Detailed description in text.
Distribution
Satellite cells are found in muscle of all vertebrates, although the frequency varies
widely depending upon location, muscle type, age and species.
It has been demonstrated that along the length of the myofiber, there is a particular
accumulation of satellite cells at the myoneural junction in both adult rat [113, 114] and
in human muscle [196]. The frequency of satellite cells is thought to be different
depending on whether the muscle is fast or slow-twitch. In fast-twitch muscle of adults,
satellite cells comprise 1 to 4 % of the total muscle nuclei (myofiber and satellite cell
nuclei), while slow-twitch muscle contains two- to three times as many [76, 179, 180].
However, the factors which govern frequency of satellite cells are poorly understood.
25
As for the effect of age on satellite cell frequency, it appears that there is a two stage
decline throughout life. During development, satellite cells are at their most abundant
stage as they are contributing to muscle growth. A first postnatal drop to the adult level
occurs at 2 months in mice and 9 years in humans [3, 39]. This reflects an increase in
myonuclei owing to fusion of satellite cell progeny during growth. Finally, a gradual
loss of satellite cells is seen as senility approaches reflecting limited proliferative
potential and eventual senescence of the satellite cell population [30].
It was thought for a long time that reliable estimates of satellite cell frequency could
only be obtained with the electron microscope. Still, although not widely adopted there
are now several staining methods for the identification of satellite cells in light
microscope preparations [49, 91, 119, 148]. Immunochemical methods are also
promising, and several antibodies have been developed, most of which react to the
neural cell adhesion molecule (N-CAM). For example, monoclonal 5.1H11 directed
against human N-CAM, binds proliferating satellite cells and myotubes in regenerating
muscle but not to quiescent satellite cells. This antibody should be useful in preparing
myogenic cells for transplant therapy of muscle disease but is less useful in animal
studies, as it is species-specific [96].
Activation and proliferation of satellite cells in response to muscle injury
Muscle satellite cells (Fig. 3 -B-) are usually quiescent but upon muscle injury they are
activated, they proliferate and finally differentiate into myoblasts (Fig. 3 -C-), which
fuse with adjacent muscle fibers or with other myoblasts (Fig. 3 -D-) to form new
multinucleated fibers (Fig. 3 -E-) [29, 81].
26
In terms of the onset of satellite cell proliferation, these have been shown to begin DNA
synthesis at about 12 to 18 h after crush injury and several hours later in the adjacent
undamaged tissue [97, 173]. In contrast to the measurement of quiescent satellite cells,
the techniques to quantify proliferating satellite cells are more reliable and include
labelling with 3H-TdR (tritiated thymidine) and bromodeoxyuridine. More recently, it
has been shown that the MIB-1 antibody raised against recombinant fragments of the Ki-
67 antigen allows retrospective evaluation of the satellite cell proliferative activity in
paraffin-embedded tissues [112]. This antibody reacts selectively with nuclei of
proliferating cells in all phases of the cell cycle [43, 75]. The satellite cells can be
distinguished from other proliferating cells such as fibroblasts, inflammatory cells or
endothelial cells because of their location beneath the basal lamina.
Upon activation after injury, satellite cells have been shown to even migrate
considerable distances within the muscle from undamaged tissue towards the injury area.
Radioautographic analysis of 3H-TdR incorporation following a focal crush injury of a
leg muscle in young rats showed that at several millimetres from the injury site the
satellite cells are stimulated to proliferate, and later migrate from the uninjured tissue
toward the crush lesion [173]. Application of myotoxic snake venom has shown that the
muscle satellite cells are capable of leaving the basal lamina [129]. In addition to
migrating within the muscle, satellite cells can also move between adjacent muscles in
the case of grafted muscle [195].
Only after 2 divisions, satellite cell progeny begin to fuse and form multinucleated
myotubes. However, satellite cell proliferation may continue for several days depending
27
on the severity of the injury
[83]
. It must be noted that different mouse strains will show
variability on the onset, duration, and effectiveness of regeneration [80, 82, 141]
Molecular events regulating muscle satellite cell activation after injury
After injury, a number of key molecular events (controlled upregulation of muscle
transcription factors and muscle specific genes) take place to regulate satellite cell
activation and differentiation during skeletal muscle regeneration. This process is
reminiscent of embryonic muscle development (previously described). Upon damage to
the myofiber (Fig. 5 -A-) the quiescent satellite cells are activated to enter the cell cycle
and proliferate, at which stage they are referred to as myogenic precursor cells (mpc) or
adult myoblasts (Fig. 5 -B-). The activation of mpc is characterised by high expression
of MRFs, Myf5 and MyoD [47].
This proliferative phase is followed by terminal differentiation (Fig. 5 -C-) and fusion of
myoblasts to damaged myofibers for repair or to each other (myotubes) for new
myofiber formation (Fig. 5 -D-). The terminal differentiation of myoblasts is
characterised by the upregulation of the MRFs Myogenin and MRF4 although MyoD is
also supposed to have a role here [65]. M-cadherin, a transmembrane protein is thought
to be an essential molecule for the specific fusion of myoblasts with each other during
muscle regeneration [111].
Finally, the repaired or new myofibers grow to resemble original myofibers (Fig. 5 -E-).
It is important to note that during the process of muscle regeneration, a subset of
myoblasts reenters the quiescent state and in this way replenish the satellite cell pool for
subsequent muscle repair (Fig. 5 -F-). The role of growth factors in this process as
28
positive or negative regulators as illustrated in figure 6, is described in detail ahead. On
the whole, there are factors which stimulate the activation of satellite cells (FGF, HGF)
and the proliferation (FGF, HGF, IGF, IL-6, LIF) and differentiation (IGF) of myoblasts
and others that inhibit the proliferation (TGF-ß1) and differentiation of myoblasts (TGF-
ß1, HGF, FGF).
Figure 5: Molecular events regulating satellite cell activation during skeletal muscle
regeneration (modified from Chargé et al., 2004) [45]. Detailed description in text.
Growth factors in regeneration
After injury, a number of growth factors or myogenic factors have an essential role in
activating the usually quiescent muscle precursor or satellite cells, which then proliferate
and differentiate to form new myotubes as described above.
29
Growth factors are proteins of fewer than 200 amino acids which are active at picomolar
concentrations. As for their origin, growth factors may be secreted from several cell
types during muscle regeneration. Indeed, they may be of autocrine origin, such as from
the satellite cells themselves, or of paracrine origin, such as from the inflammatory cells
(mostly macrophages) or the motor nerve [90] (Fig. 6). The signal from the different
growth factors is mediated by specific growth factor receptors, many of which are
tyrosine kinase receptors.
It is thought that two types of growth factors have to act upon the quiescent satellite cells
before sustained proliferation is initiated: 1) a competence growth factor to move the
cells from the quiescent G0 state into the G1 phase of the cell cycle; 2) a progression
growth factor to regulate the progression through G1 to the DNA synthesis phase and
thus sustain proliferation [81, 150]. FGF and PDGF are the most well known examples
of competence factors whereas the IGF family are a good example of progression factors
[133]. Apart from their role in activating the satellite cells, growth factors also stimulate
the chemotaxis of further satellite cells to the injured area.
A large body of evidence supports the role of individual growth factors/cytokines such
as IGF-1, FGF-2, PDGF, HGF, NGF, TGF-ß1, EGF, LIF, IL-4, IL-6, IL-18 and HARP
during muscle regeneration [4, 98, 109, 135, 193]. Of these trophic substances FGF-2,
IGF-I, HGF and TGF-ß1 are thought to be key regulators in the chemotaxis and
activation of satellite cells [25, 90, 123, 177, 201].
The actions of the most important myogenic factors will be dealt with in more detail in
what follows ahead.
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- , 2005, New Approaches for the Treatment of Muscle Injuries, Hamburg, Bedey Media GmbH, https://www.diplom.de/document/224046
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