Transcript Satellite cells

Molecular Exercise Physiology
Myogenesis and Satellite Cells
Presentation 9
Henning Wackerhage
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.
Myogenesis and satellite cells
Part 1
Myogenesis
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
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.
Myogenesis
Proliferation (cells
divide and increase
in number)
Somite cells
Fusion
Myoblasts
Muscle
fibres
During myogenesis, undifferentiated cells first differentiate into muscle
precursor cells (myoblasts) and then fuse and become myotubes and
then muscle fibres.
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.
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
knocked
out
Viable
Phenotype
Myoblasts
Myotubes
Role of
myogenic
protein
MyoD
Yes
+
+
?
Myf5
Yes
+
+
?
MyoD&
Myf5
No
-
-
Required for
myoblast formation
+
-
Required for myoblast
differentiation into
muscle
Myogenin No
Myogenesis
Primary MRF’s regulate
determination
MyoD or Myf-5
Somite cells
Secondary MRF’s regulate fusion of
myoblasts and terminal differentiation
Myogenin
MRF4
Proliferation
Fusion
Myoblasts
Muscle fibres
Myogenic regulatory factors (MRFs) regulate the “muscle making”
process. They appear at different times during muscle development.
Myogenesis
(b)
(a)
E-protein
Transcription of
muscle genes
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.
Task
Do myogenic factors change in response to exercise? Find out!
Myogenesis and satellite cells
Part 1
Satellite cells and hypertrophy
Tissue can grow in two ways
Hyperplasia
(more cells)
Nucleus
Hypertrophy
(larger cells)
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”.
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.
Two possibilities
Hypertrophy with
DNA dilution (only
protein synthesis)?
Mechanism for
increase in
nuclear number
DNA-to muscle
volume is kept
constant during
hypertrophy
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.
Satellite cells
Satellite cells:
• Were
discovered
via
electron
microscopy
by
Mauro
(1961)
in
frog
myofibres.
Satellite cell • are active in young,
growing
muscle
and
quiescent
in
older
muscles (Schultz 1976).
Myonucleus • Donate
myonuclei
into
growing
muscle
fibres
(Moss and Leblond 1971).
Kadi et al. (1999)
Basal lamina
Satellite cells
Satellite cell
Plasmalemma
Myonucleus
Figure: Location of a torpedo-shaped
plasmalemma and basal lamina.
satellite
cell
between
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)
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).
Parallel increase in muscle volume and nuclei number
C control; PL power lifter.
Kadi et al. (1999)
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.
Satellite cells and hypertrophy
•
•
EDL muscle mass
(% compared to control)
•
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
Irr.
Irr. Irradiation; Abl. Ablation.
Abl.
Irr.+Abl. Control
Rosenblatt et al. (1994)
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.
Role of satellite cells in hypertrophy
Satellite cell
proliferation and
differentation
Hypertrophy
stimulus
Fusion of some
satellite cell with
muscle fibre
Satellite cell (mononucleated, early developmental age)
Nucleus donated by satellite cell
Task
What happens when muscle fibres atrophy? Do they lose
myonuclei and if how?
The End