Molecular Exercise Physiology Myogenesis and Satellite Cells Presentation 9 Henning Wackerhage

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Molecular Exercise PhysiologyMyogenesis and
Satellite Cells Presentation 9Henning Wackerhage
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Learning outcomes

• At the end of this lecture, you should be able
  to
• Describe how mononucleated, undifferentiated
  cells differentiate and fuse to turn into adult
  skeletal muscle fibres.
• Explain how this process is regulated by myogenic
  regulatory factors.
• Explain what satellite cells are and what their
  function is.

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Myogenesis and satellite cellsPart 1Myogenesis
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Adult skeletal muscle
Adult skeletal muscle fibres have hundreds to
thousands of nuclei that are located at the
periphery of the fibres.
Nucleus
Longitudinal section
Cross section
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Myogenesis muscle development
Muscle fibres can be several tens of centimetres
long. Using the values found by Tseng et al. in
adult rat fibres, a 10 cm long skeletal muscle
fibre contains between 4000 and 12000 nuclei
with a higher nuclear density found in type 1
fibres (Tseng et al., 1994). Research into
myogenesis is addressing the question how do
multinuclear muscle fibres form during
development? The answer is simple Muscle
precursor cells, so-called myoblasts align and
fuse into multinuclear myotubes. Myogenesis is
regulated by myogenic regulatory factors (MRFs).
MRFs are transcription factors that appear during
development. When bound DNA they increase the
expression of proteins that will turn a
non-muscle cell first into a cell expressing
muscle genes (myoblast) and other MRFs will then
promote the fusion of myoblasts into myotubes and
finally fully differentiated muscle fibres.
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Myogenesis
Proliferation (cells divide and increase in
number)
During myogenesis, undifferentiated cells first
differentiate into muscle precursor cells
(myoblasts) and then fuse and become myotubes and
then muscle fibres.
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MRFs regulate myogenesis
Breakthrough finding Davis et al. (1987) knew
that 5-azacytidine treatment converted
fibroblasts into muscle cells. Thus,
5-azacytidine treatment must have induced factors
that regulate the conversion from fibroblasts to
muscle. The strategy of Davis et al. was to
search for mRNA that was present in muscle cells
but not in fibroblasts. They identified several
candidates that were named MyoA, MyoD and MyoH.
In a second experiment, they transfected
fibroblasts with MyoA, MyoD and MyoH. Only
fibroblasts that were transfected with MyoD
expressed the muscle protein myosin. Thus, MyoD
must be a muscle maker, a myogenic regulatory
factor (MRF). Subsequently, other researchers
identified mrf4, myogenin and myf-5 as MRFs using
similar strategies.
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Effect of MRF knockout on myogenesis
In subsequent experiments, MRFs were knocked
out in mice in order to understand their
function during myogenesis. Surprisingly, a
knockout of MyoD or Myf5 did not have an effect.
Only knocking out both prevents myogenesis,
suggesting that MyoD and Myf5 are redundant.
Please analyse the following table.
Gene Phenotype Role of knocked
myogenic out Viable Myoblasts Myotubes p
rotein MyoD Yes ? Myf5
Yes ? MyoD No - - Required
for Myf5 myoblast formation Myogenin
No - Required for myoblast differentia
tion into muscle
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Myogenesis
Primary MRFs regulate determination
Secondary MRFs regulate fusion of myoblasts and
terminal differentiation
Myogenin
MRF4
MyoD or Myf-5
Fusion
Muscle fibres
Myogenic regulatory factors (MRFs) regulate the
muscle making process. They appear at different
times during muscle development.
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Myogenesis
(b)
(a)
Transcription of muscle genes
E-protein
MRF
CANNTG
E-box
(a) MRFs dimerize with E-proteins and bind a
CANNTG DNA sequence which is termed E-box. MRF
DNA binding is essential for the expression of
muscle genes and other genes that are involved in
muscle making. (b) Structurally, MRFs are
helix-loop-helix (HLH) proteins. HLH domains are
DNA-binding domains which is shown below.
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Task
Do myogenic factors change in response to
exercise? Find out!
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Myogenesis and satellite cellsPart 1Satellite
cells and hypertrophy
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Tissue can grow in two ways
Tissues can grow in two ways Cells can double
their nuclei/DNA and contents in a process called
cell cycle and then split. This growth is called
hyperplasia and is the most common form of muscle
growth. Cells can also grow in size and this
process is called hypertrophy. However, cellular
hypertrophy is limited because the DNA
concentration within a cell with one nucleus will
be diluted.
Hyperplasia (more cells)
Nucleus
Hypertrophy (larger cells)
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Satellite cells
Skeletal muscle can hypertrophy or atrophy. The
nuclei within a muscle fibre, however, are
post-mitotic and cannot divide anymore. Assume
that the volume of a fibre increases by 25 . If
the nuclear number would be the same then the DNA
(which is the main component of nuclei) would be
diluted and the capacity for transcription would
be decrease in a growth situation. Thus, does a
mechanism exist that keeps the nucleus-to-volume
ration (the so-called myonuclear domain)
constant? The next slide illustrates the problem.
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Two possibilities
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Satellite cells
Several lines of research have shown that nuclear
numbers increase or decrease in parallel with the
volume of a fibre. Thus, a mechanism must exist
that involves nuclei other than The new nuclei
originate from so-called satellite cells these
cells are mononucleated reserve muscle cells
that lie on the surface of muscle fibres and are
capable of proliferating (they increase in
number), differentiating (develop further towards
mature muscle) and fusion with muscle fibres.
Satellite cells appear to be mononuclear muscle
cells that remain at an early developmental age.
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Satellite cells

• Satellite cells
• Were discovered via electron microscopy by Mauro
  (1961) in frog myofibres.
• are active in young, growing muscle and quiescent
  in older muscles (Schultz 1976).
• Donate myonuclei into growing muscle fibres (Moss
  and Leblond 1971).

Satellite cell
Myonucleus
Kadi et al. (1999)
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Satellite cells
Basal lamina
Satellite cell
Plasmalemma
Myonucleus
Figure Location of a torpedo-shaped satellite
cell between plasmalemma and basal lamina.
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Satellite cells
Here, a human satellite cell is shown by electron
microscopy. They are located inside the basal
lamina (arrowheads) and outside the sarcolemma
(arrows) and an independent cytoplasm. Bar, 1 µm
(Sinha-Hikim et al. 2002)
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Satellite cells
The next slide shows that the number of nuclei
per cross-section of a muscle fibre is maintained
in athletes that achieve hypertrophy mainly due
to resistance training. This is indirect
evidence for an increase in nuclear numbers in
response to resistance training (although no
evidence for the action of satellite cells).
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Parallel increase in muscle volume and nuclei
number
C control PL power lifter.
Kadi et al. (1999)
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Satellite cells
The next slide shows the results of a key
experiment. Rosenblatt et al. irradiated muscles
which is known to stop proliferation. It also
stopped the proliferation of satellite
cells. They then applied synergist ablation as a
hypertrophy stimulus. In this model a synergist
is removed and thus the remaining muscle is
overloaded and hypertropies. The most important
finding is that when irradiation and ablation
were applied together, no hypertrophy resulted.
The findings suggest that satellite cells are
essential for skeletal muscle hypertrophy.
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Satellite cells and hypertrophy

• Irradiation blocks (satellite) cell division.
• No hypertrophy when irradiated despite
  hypertrophy stimulus in this model.
• However, muscle fibres can grow without
  proliferation in culture.

120 115 110 105 100 95

EDL muscle mass ( compared to control)
Irr. Abl. Irr.Abl. Control
Irr. Irradiation Abl. Ablation.
Rosenblatt et al. (1994)
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Satellite cells
Together, the research on satellite cell
suggests Growth stimuli such as IGF-1 activate
and many atrophy stimuli such as myostatin
inhibit the proliferation and differentation of
satellite cells. Satellite cells then fuse with
their muscle fibres. Due to this mechanism, the
myonuclear domain (the ratio between nucleus and
cytoplasmic volume) is maintained.
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Role of satellite cells in hypertrophy
Satellite cell proliferation and differentation
Hypertrophy stimulus
Satellite cell (mononucleated, early
developmental age) Nucleus donated by satellite
cell
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Task
What happens when muscle fibres atrophy? Do they
lose myonuclei and if how?
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The End