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. Learning outcomes
Atrophy Part 1 Protein breakdown (proteolysis)
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. 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. 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. Skeletal muscle atrophy
Calpain Ca 2+ ↑ Z-disc Ub 26S Proteasome (breaks down protein) Peptides Ub E1 ATP E2 E3 Ub Protein Sarcomere Skeletal muscle proteolysis 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. (1) (2) E2 Ub
26S Proteasome 20S Proteasome (proteolysis) Base (ATPases, unfolding, translocation) Lid (Ubiquitin binding)
E3 ubiquitin ligases 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. Days
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!).
E3 ubiquitin ligases determine proteolysis rate 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. Ub 26S Proteasome Peptides Ub E1 ATP E2 E3 Ub Protein E3 ligases connect ubiqitins to proteins selected for breakdown
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. Cathepsin L Cathepsin Lysosome Peptides Cathepsin
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. Problem!
Atrophy Part 2 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. Starvation Question: Was David Blaine’s stunt risky?
Elia M: Hunger disease. Clin. Nutr. (19): , 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. 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
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).
Starvation Body mass index (kg/m 2 ) Protein oxidation (of basic metabolic rate) 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. (Elia 2000)
Subjects that are most obese survive for the longest time due to their fat reserves. Starvation Available energy Protein kcal (21%) Fat75200 (79%) Total95600 Survival64 days at 1500 kcal/day Available energy Protein (6%) Fat (79%) Total Survival days at kcal/day Lean 70 kg man (13% fat) 12.2 kg protein; 4.6 kg available 9 kg fat, 8 kg available Obese 140 kg man (44% fat) 15.7 kg protein; 8.1 kg available 61.5 kg fat, 60.5 kg available
Task: Assume a 90 kg man with 13.6 kg protein (6 kg available) and 25.2 kg fat (24.2) available. Assume a basal metabolic rate of 1800 kcal/day and calculate likely survival time during starvation 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! Gene regulation during starvation
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.
Atrophy Part 3 Cachexia
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 (TNF) which is the name used today. Injecting TNF into rats causes muscle wasting. The following slide gives an overview over the effects of TNFon skeletal muscle.
IBIB NF-B TNF signalling and cachexia IBIB IKK Degradation TNF Ubiquitin, proteasome? MyoD P NF-B TNF-receptor TNF via its receptors activates the nuclear factor-B (NF-B) pathway. TNFa binding to its receptor causes and activation of IK kinase (IKK) which phosphorylates the inhibitor of B (IB). IB normally binds to NF-B. When phosphorylated, IkB detaches from NF-B and NF-B translocates into the nucleus and binds to B enhancers. This increases the expression of the proteolytic machinery and decreases MyoD. However, the actions of TNF and NF-B are not fully understood P
Cachexia TNF 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 TNF twofold after a Marathon race (Pedersen, 2000), indicating that increases in pro-inflammatory cytokines are not limited to disease.
Myostatin overexpression causes cachexia A systemic increase in myostatin leads to a skeletal muscle atrophy and fat loss in adult mice (Zimmers et al. 2002). In addition, myostatin is another candidate for cachexia. Increased myostatin levels had been demonstrated in HIV patients (Gonzales- Cadavid et al. 1998).
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 TNF, are increased an are likely to increase protein breakdown and inhibit myogenesis.
Atrophy Part 5 Ageing sarcopenia
Ageing sarcopenia The primary problem in ageing sarcopenia appears to be a loss of - 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). Young Old -motor neurones Muscle fibres 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.
Ageing sarcopenia s 70s Age (decade) Body composition (%) Non-muscle fat-free mass Fat Muscle Balagopal et al. (1997) 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.
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 TNF 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.
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 -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.
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.