Molecular Exercise Physiology Myogenesis and Satellite Cells Presentation 9 Henning Wackerhage.
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Transcript of Molecular Exercise Physiology Myogenesis and Satellite Cells Presentation 9 Henning Wackerhage.
Molecular Exercise PhysiologyMyogenesis and Satellite Cells
Presentation 9Henning Wackerhage
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.
Learning outcomes
Myogenesis and satellite cellsPart 1
Myogenesis
Adult skeletal muscle fibres have hundreds to thousands of nuclei that are located at the periphery of the fibres.
Adult skeletal muscle
Nucleus
Longitudinal section Cross section
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 = muscle development
Somite cells Myoblasts
Muscle fibres
Fusion
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.
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.
Gene Phenotype Role ofknocked myogenicout Viable Myoblasts Myotubes protein
MyoD Yes + + ?
Myf5 Yes + + ?
MyoD& No - - Required forMyf5 myoblast formation
Myogenin No + - Required for myoblastdifferentiation into muscle
Effect of MRF knockout on myogenesisIn 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.
Somite cells Myoblasts
Muscle fibres
Fusion
Myogenesis
Proliferation
MyoD or Myf-5 Myogenin MRF4
Primary MRF’s regulatedetermination
Secondary MRF’s regulate fusion of myoblasts and terminal differentiation
Myogenic regulatory factors (MRFs) regulate the “muscle making” process. They appear at different times during muscle development.
Myogenesis
(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.
MRF
E-protein
CANNTG
Transcription of muscle genes
(a) (b)
E-box
Task
Do myogenic factors change in response to exercise? Find out!
Myogenesis and satellite cellsPart 1
Satellite cells and hypertrophy
Tissue can grow in two ways
Hyperplasia (more cells)
Hypertrophy (larger cells)
Nucleus
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”.
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.
Satellite cells
Two possibilities
Hypertrophy with DNA dilution (only protein synthesis)?
DNA-to muscle volume is kept constant during
hypertrophy
Mechanism for increase in
nuclear number
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 cell
Myonucleus
Kadi et al. (1999)
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 cells
Basal lamina
Plasmalemma
Myonucleus
Satellite cell
Satellite cells
Figure: Location of a torpedo-shaped satellite cell between plasmalemma and basal lamina.
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)
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).
Satellite cells
Kadi et al. (1999)
C control; PL power lifter.
Parallel increase in muscle volume and nuclei number
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
• Irradiation blocks (satellite) cell division.• No hypertrophy when irradiated despite hypertrophy stimulus in
this model.• However, muscle fibres can grow without proliferation in
culture.
Rosenblatt et al. (1994)
Satellite cells and hypertrophy
120
115
110
105
100
95
Irr. Abl. Irr.+Abl. Control
ED
L m
usc
le m
ass
(%
com
pare
d t
o c
on
trol)
Irr. Irradiation; Abl. Ablation.
*
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.
Satellite cells
Role of satellite cells in hypertrophy
Fusion of some satellite cell with
muscle fibre
Satellite cell (mononucleated, early developmental age)
Nucleus donated by satellite cell
Satellite cell proliferation and
differentation
Hypertrophy stimulus
Task
What happens when muscle fibres atrophy? Do they lose myonuclei and if how?
The End