1. Molecular Exercise Physiology Myogenesis and Satellite Cells Presentation 9 Henning Wackerhage

2. 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

3. Myogenesis and satellite cells Part 1 Myogenesis

4. Adult skeletal muscle fibres have hundreds to thousands of nuclei that are located at the periphery of the fibres. Adult skeletal muscle Nucleus Longitudinal sectionCross section

5. 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

6. 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.

7. 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.

8. GenePhenotypeRole of knocked myogenic out ViableMyoblastsMyotubesprotein MyoD Yes++? Myf5 Yes++? MyoD& No--Required for Myf5myoblast formation Myogenin No+-Required for myoblast differentiation into muscle 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.

9. Somite cells Myoblasts Muscle fibres Fusion Myogenesis Proliferation MyoD or Myf-5 MyogeninMRF4 Primary MRF’s regulate determination 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.

10. 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

11. Task Do myogenic factors change in response to exercise? Find out!

12. Myogenesis and satellite cells Part 1 Satellite cells and hypertrophy

13. 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”.

14. 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

15. Two possibilities Hypertrophy with DNA dilution (only protein synthesis)? DNA-to muscle volume is kept constant during hypertrophy Mechanism for increase in nuclear number

16. 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

17. 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

18. Basal lamina Plasmalemma Myonucleus Satellite cell Satellite cells Figure: Location of a torpedo-shaped satellite cell between plasmalemma and basal lamina.

19. 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)

20. 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

21. Kadi et al. (1999) C control; PL power lifter. Parallel increase in muscle volume and nuclei number

22. 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

23. 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 EDL muscle mass (% compared to control) Irr. Irradiation; Abl. Ablation. *

24. 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

25. 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

26. Task What happens when muscle fibres atrophy? Do they lose myonuclei and if how?

27. The End