1. Molecular Exercise Physiology Atrophy Presentation 10 Henning Wackerhage

2. Learning outcomes
At the end of this presentation you should be able to:
Describe the skeletal muscle protein breakdown systems and explain how they disassemble a sarcomere.
Compare and contrast the changes that lead to skeletal muscle atrophy during starvation, cachexia and ageing sarcopenia.

3. Atrophy Part 1 Protein breakdown (proteolysis)

4. Skeletal muscle atrophy
Proteins largely determine the size of the muscle. Thus, muscle growth and atrophy is a question of protein content. The protein concentrations depends on both protein synthesis and protein breakdown:
[Protein] = protein synthesis – protein breakdown.
A decreasing protein concentration can occur due:
a) A reduction in protein synthesis alone;
b) An increase in protein breakdown alone;
c) A reduction in protein synthesis and increase in protein breakdown;
d) An increase in protein synthesis and even higher increase in protein breakdown.

5. Skeletal muscle atrophy
Skeletal muscle atrophy can result from a large number of causes:
Immobilisation (space flight, cast immobilisation, physical inactivity);
Disease-related (cancer, HIV, rheumatoid arthritis, renal failure, burns, sepsis);
Drug-related (glucocorticoids);
Inadequate nutritional intake or digestion (starvation, protein malnutrition, impaired digestion);
Ageing.
The following slide gives an overview over the factors involved.

6. Food/protein intake↓ (anorexia, starvation)
Ageing factors↑
Food/protein intake↓ (anorexia, starvation)
Apoptosis/necrosis↑
Proinflammatory cytokines↑ (TNFa, interleukin-1, interleukin-6)
Impaired digestion
Expression↓/inhibition of transcription/translational components
Catabolic hormones and factors↑ (glucocorticoids, thyroid hormones, myostatin)
Expression↑/activation of proteolytic systems
Anabolic hormones and factors↓ (growth hormone, IGF-1, androgens, clenbuterol)
Factors derived from pathological tissues (proteolysis-inducing factor, PIF)
Unloading, innervation↓, inactivity
Inheritance

7. Skeletal muscle atrophy
The degradation of a muscle sarcomere is likely to be a two-step process:
1) First, calpains disassemble the sarcomere at the Z-line (by digesting Z-line proteins such as nebulin and fodrin) and break it up in smaller pieces (Huang and Forsberg 1998).
2) In the second step, myosin, actin and other proteins are then digested by the ATP-ubiquitin-dependent 26S proteasome which can not digest connected proteins (Solomon and Goldberg 1996).
Finally, some proteins are also degraded by cathepsins, which are located in organelles that are called lysosomes. In addition, proteases from mast cells (immune cells) can also contribute to skeletal muscle breakdown.
Protein breakdown by calpains and the ATP-ubiquitin-dependent 26S proteasome is shown on the next slide.

8. 26S Proteasome (breaks down protein)
Skeletal muscle proteolysis
ATP
(2)
Sarcomere Ub E1 E1
(1) Ub E2
Protein Ub E2 E3
Z-disc
Calpain
Ca2+↑ Ub
Peptides Ub
26S Proteasome (breaks down protein)
Overview over skeletal muscle proteolysis. (1) First, the sarcomere is broken up at the Z-disc via calcium dependent proteases named calpains. After that, proteins are ubiquitinated (attached to the protein ubiquitin) in an ATP-dependent reaction involving ubiquitin ligases (E1-E3). Ubiquitinated proteins are then digested by the 26S proteasome.

9. Base (ATPases, unfolding,
26S Proteasome
Lid (Ubiquitin
binding)
20S Proteasome (proteolysis)
a b b a
Base (ATPases, unfolding,
translocation)

10. E3 ubiquitin ligases Days
Bodine et al. (2001) used DNA microarray analysis of various atrophy states in order to determine genes that change their expression during atrophy. They identified two E3 ligases, MuRF1 and MAFbx that are increasingly expressed during atrophy (see figure; mRNA for MuRF1 and MAFbx has been measured with RT-PCR to confirm microarray results). E3 ligases transfer a ubiquitin to proteins selected for degradation by the 26S proteasome.

11. E3 ubiquitin ligases affect proteolysis rate
Bodine et al. (2001) also showed that overexpression of MAFbx causes atrophy. The reseachers also knocked out MAFbx and MuRF1 and this led to a significant limitation in muscle loss in response to denervation (see figure). Thus, the concentration of E3 ligases such as MAFbx and MuRF1 determines the protein breakdown rate and net protein changes in this model (it can differ!).

12. E3 ubiquitin ligases determine proteolysis rate
ATP E1 E1 Ub E2 Ub
E3 ligases connect ubiqitins to proteins selected for breakdown
Protein E3 Ub Ub
Peptides Ub
26S Proteasome
Thus, it appears that the upregulation of E3 ligases such as MAFbx and MuRF1 is sometimes a mechanism for increasing the rate of protein breakdown, at least in skeletal muscle. Jagoe et al. (2002) found that atrogin-1 (MAFbx), another E3 ligase, increased in respond to food deprivation in skeletal muscle.

13. Cathepsin L
Cathepsin
Cathepsin
Peptides
Lysosome
A third class of a proteases are called cathepsins is a protease that is located in organelles called lysosomes. Cathepsin L was discovered as a mRNA and protein that was induced in muscle wasting conditions such as sepsis, cancer or dexamethasone treatment. The left figure shows that cathepsin L is induced by dexamethasone, a synthetic glucocorticoid that is known to induce muscle atrophy (Deval et al. 2001). Cathepsin L mRNA also increases in response to unloading.

14. Problem!
Quite often, parts of the protein breakdown machinery expression increase whereas muscle protein breakdown itself decreases. E.g. the E3 ubiquitin ligase atrogin-1 (MAFbx) increases during starvation (Jagoe et al. 2002) but protein breakdown is actually initially decreased in response to starvation until it rises during the later stages of starvation (Emery et al. 1986).
Thus, parts of the protein breakdown machinery are not good markers for protein breakdown. You have to measure protein breakdown directly.

15. Atrophy Part 2 Starvation

16. Starvation
Starvation is still a problem in parts of the developing world and it was somewhat trivialised by David Blaine, who earned his money by starving for 44 days in a plexiglas box in London.
Question: Was David Blaine’s stunt risky?

17. Starvation Elia M: Hunger disease. Clin. Nutr. (19):379-386, 2000:
Some invertebrates may lose as much as 95 % of body weight before they die from starvation. Mammals die often with losing less than 50%.
Nine out of ten Irish hunger strikers died between 57 and 73 days of starvation losing about 40% of body weight.
The factor that determines survival time during starvation is the body’s total energy reserves. Grossly obese subjects can lose 65-80% of total body weight.
Some human beings have starved for over 100 days and one obese subject has starved for almost 382 days (Stewart and Fleming (1973).
Answer: David Blaine’s 44 days showcase starvation is not life threatening esp. because he had put on weight before.

18. Starvation
Skeletal muscle is lost during starvation depending predominantly on the amounts of energy in the body at the beginning of starvation and on the length of starvation. Generally, energy is preferably derived from carbohydrates as fat whereas muscle and to a greater extent the brain are conserved (see below):
Carbohydrates > fat > muscle protein > brain fat, protein

19. Starvation and protein synthesis
Fasting reduces protein synthesis in skeletal muscle and feeding doubles protein-synthesis in fasted men (Rennie et al. 1982).
Fasting in rats initially decreases protein breakdown but after prolonged fasting protein breakdown is increased (Emery et al. 1986).
The effect of fasting and refeeding depends on the length of the fasting period and the amount of energy available from other sources (adipose tissue, glycogen).

20. (of basic metabolic rate)
Starvation 40 30 20 10
(of basic metabolic rate)
Protein oxidation
Body mass index (kg/m2)
(Elia 2000)
The effects of initial body mass index on the contribution of protein oxidation to basal metabolic rate during starvation. Conclusion: Obese subjects also survive longer because they oxidise less of the functional protein initially.

21. Starvation
Subjects that are most obese survive for the longest time due to their fat reserves.
Lean 70 kg man (13% fat)
12.2 kg protein;
4.6 kg available
9 kg fat, 8 kg available
Available energy
Protein kcal (21%)
Fat (79%)
Total
Survival 64 days at kcal/day
Obese 140 kg man (44% fat)
15.7 kg protein;
8.1 kg available
61.5 kg fat, 60.5 kg available
Available energy
Protein (6%)
Fat (79%)
Total
Survival days at kcal/day

22. Starvation
Task: Assume a 90 kg man with 13.6 kg protein (6 kg available) and kg fat (24.2) available. Assume a basal metabolic rate of 1800 kcal/day and calculate likely survival time during starvation ?

23. Gene regulation during starvation
Jagoe et al. FASEB J. 16: , 2002:
Decrease in total RNA (decrease in ribosome content);
Ubiquitin-proteasome mRNA increases: greater capacity for muscle protein breakdown.
Carbohydrate catabolism enzymes decrease but no upregulation of fat metabolism enzymes: Preservation of the limited glycogen reserves.
Myosin heavy chain and collagen expression goes down.
Apoptosis markers increase.
4E-BP1 increases: Increased inhibition of translation? Unclear!

24. Starvation: conclusion
Starvation leads to a decrease in protein synthesis and protein breakdown with a net loss of protein.
Survival and usage of muscle protein depends on the duration of the starvation period and on the amount of fat available.
The more fat is used up, the more muscle protein is oxidised and thus the rate of net protein breakdown is highest near death due to starvation.

25. Atrophy Part 3 Cachexia

26. Cachexia
In cachexia, both muscle and fat degrade and the loss of muscle can not be reversed by increasing nutritional intake.
Cachexia is derived from kakos for "bad" and hexis for "condition”. Thus, cachexia is bad condition.
Norton et al. (1985) linked the circulation of tumour-bearing rats with normal rats. Both rats displayed cachexia. The experiment showed that a circulating agent was released from the tumour and caused cachexia.
At the beginning of the 1980s, Cerami’s research group identified a molecule that was responsible for muscle wasting and was named “cachectin”. Cachectin was shown to be identical with tumour necrosis factor a (TNFa) which is the name used today. Injecting TNFa into rats causes muscle wasting. The following slide gives an overview over the effects of TNFa on skeletal muscle.

27. Ubiquitin, proteasome? MyoD
TNFa signalling and cachexia
TNFa
TNF-receptor
Ubiquitin, proteasome? MyoD
IKK P
IkB
NF-kB
NF-kB P
IkB
Degradation
TNFa via its receptors activates the nuclear factor-kB (NF-kB) pathway. TNFa binding to its receptor causes and activation of IK kinase (IKK) which phosphorylates the inhibitor of kB (IkB). IkB normally binds to NF-kB. When phosphorylated, IkB detaches from NF-kB and NF-kB translocates into the nucleus and binds to kB enhancers. This increases the expression of the proteolytic machinery and decreases MyoD. However, the actions of TNFa and NF-kB are not fully understood

28. Cachexia
TNFa is not the only pro-inflammatory cytokine that is increased in disease states associated with a loss of muscle mass and that causes muscle atrophy. Interleukin-1 and interleukin-6 also cause muscle wasting when injected and are also increased in several disease states. In addition, a so-called proteolysis-inducing factor (PIF) is released in several forms of cancer.
Disease states with a rise in pro-inflammatory cytokines are: sepsis (Cohen, 2002), rheumatoid arthritis (Walsmith & Roubenoff, 2002), Aids/HIV (Emilie et al., 1994), burns (Schwacha & Chaudry, 2002), pancreatitis (Makhija & Kingsnorth, 2002), heart failure (Anker et al., 1999), renal failure (Klahr & Morrissey, 2003) and some forms of cancer (Tisdale, 2001). A moderate increase in pro-inflammatory cytokines is also often seen in ageing (Roubenoff, 2003).
In addition, pro-inflammatory cytokines such as interleukin-6 increase up to 100-fold and TNFa twofold after a Marathon race (Pedersen, 2000), indicating that increases in pro-inflammatory cytokines are not limited to disease.

29. Myostatin overexpression causes cachexia
In addition, myostatin is another candidate for cachexia. Increased myostatin levels had been demonstrated in HIV patients (Gonzales-Cadavid et al. 1998).
A systemic increase in myostatin leads to a skeletal muscle atrophy and fat loss in adult mice (Zimmers et al. 2002).

30. Cachexia: conclusion
In cachexia, both muscle and fat degrade and the loss of muscle can not be reversed by increasing nutritional intake.
Muscle protein breakdown is increased at least in some cachexia-inducing disease states. In some disease states, such as some forms of cancer, protein loss can be rapid and extreme.
Several pro-inflammatory cytokines, most notably TNFa, are increased an are likely to increase protein breakdown and inhibit myogenesis.

31. Atrophy Part 5 Ageing sarcopenia

32. Ageing sarcopenia
The primary problem in ageing sarcopenia appears to be a loss of a-motor neurones (Tomlinson & Irving, 1977;Kawamura et al., 1977). As a result, almost half of the muscle fibres are lost from the age of 20 to the age of 80, at least in the vastus lateralis (Lexell et al. 1988).
a-motor neurones
Muscle fibres
Young
Old
Ageing sarcopenia is a very slow process. Lexell et al.’s (1988) data suggest that  12 fibres out of half a million are lost daily from the age of 20 to the age of 80 years. Similarly, a hypothetical loss of 10 kg of muscle mass over 40 years equates to a daily muscle loss of ≈ 0.7 g. Thus, the net muscle changes are very hard to detect.

33. Ageing sarcopenia
Ageing sarcopenia leads to a change in body composition. The relative contribution of fat and non-muscle fat free muscle increases whereas the contribution of muscle decreases.
100 75 50 25
Non-muscle fat-free mass
Fat
Muscle
Body composition (%)
20s s
Age (decade)
Balagopal et al. (1997)

34. Other ageing sarcopenia changes
Type 2 fibres atrophy and the percentage of type 2 fibres decreases (Lexell et al. 1988).
Basal protein synthesis and degradation are probably unchanged (Dorrens & Rennie, 2003). However, the response to resistance training and nutrition is likely to be different.
The slow elimination of fibres and myonuclei is likely due to apoptosis, at least in rats (Dirks & Leeuwenburgh, 2002;Pollack et al., 2002).
Aging causes cell death and functional changes in the neuroendocrine system (Rehman & Masson, 2001) and this affects the growth environment of the muscle.
The pulse amplitude of growth hormone secretion (Finkelstein et al., 1972) and systemic IGF-1 concentrations decrease with aging (Copeland et al., 1990).
In addition, old age is often associated with a low-grade inflammation as demonstrated by higher levels of cytokines such as TNFa and IL-6 (Bruunsgaard et al., 2001) and an “inflammation theory of aging” has been proposed (Chung et al., 2001).
Serum myostatin is higher in older men and women than in young (Yarasheski et al., 2002), although there is a high variation.

35. Ageing sarcopenia: conclusion
Ageing sarcopenia is a very slow process and it is very difficult to detect net changes.
The primary cause appears to be a loss of a-motor neurones which is followed by a loss of muscle fibres.
Basal muscle protein synthesis and breakdown appear to be unchanged.
Many other changes could contribute to the net loss of muscle mass: decreased growth hormone, IGF-1, increased myostatin and pro-inflammatory cytokines.

36. Task
You have a patient with cachexia and with severe ageing sarcopenia and you are asked to develop treatments. Name at least three strategies for treating both conditions.
Second, atrophy can also result from inactivity. Find out more about the cause.

37. The End