Файл: Extracellular and Intracellular Signaling (книга).pdf

mTORc1 changes. Collectively, these data show that while signaling through these anabolic pathways is largely linear (i.e. ligand receptor - kinase-kinase- kinase - kinase-kinase - kinase - e ect) during development or specific disease states, complex non-linear signaling and cross-talk between what were previously thought of as distinct pathways occurs in adults subject to exercise.

Currently it seems that muscles control proteostasis in response to muscular activity largely from within. From the limited data available, it seems likely that physical deformation of the cell (i.e. mechanotransduction) and biochemical perturbations (i.e. chemotransduction) are key upstream regulators of the phosphoproteins regulating muscle proteostasis. Mechanotransduction is probably sensed though physical transmembrane links (i.e. the attachment complex) between the extracellular matrix and the actin cytoskeleton to which many of the anabolic signals may be physically tethered. However, titin, a major structural component of the contractile apparatus, has also been shown to be a stretch-activated kinase. Following stretch activation of the kinase substrates such as nbr1 and p62 are phosphorylated to create binding sites. Notably NFkB signaling can be activated via p62, although the relevance of this in human muscle remains to be established. The E3 ligase MuRF2 can also be activated by p62 and upon activation causes serum response factor (SRF) to translocate to the sarcoplasm from the nucleus. SRF acts as a transcription factor and participates in the expression of muscle genes, thus titin may modulate long-term muscle gene expression via modulation of SRF, which likely accounts for the presence of hereditary myopathy with early respiratory failure in individuals with mutations in titin kinase. Despite the promise of titin as a mechanosensor within muscle, this mechanism does not currently explain the activation of known anabolic signals in response to exercise.

On the basis of chemotransduction, muscular activity has been associated with many metabolic changes. As examples, muscular activity generates alterations in ion movements via opening of stretch-activated channels (SAC) in the plasma membrane and fluctuations in SR-derived [Ca21]i to facilitate contraction. Also increased ATP turnover during contraction produces metabolites, which are also able to modulate cellular signaling processes. Furthermore, as a by-product of increased ATP turnover, reactive oxygen species (ROS) are generated, which function to activate redox-sensitive pathways such as NFkB (see Section 5.2.2.4). Finally, production of lipid second messengers derived from the plasma membrane, which can be damaged during contraction, regulates cell signaling processes.

While the precise control of muscle proteostasis in human muscle subject to exercise remains elusive, there are several lines of evidence from in vitro studies that the anabolic signals observed are mechano-/chemosensitive (see Figure 5.2 for summary scheme). For example, inhibition of extracellular Ca21 influx through SAC inhibits mechanical stretch-dependent signaling to p70S6K1, suggesting that extracellular Ca21 is required for signaling to mTORc1. Likewise, disruption of the actin cytoskeleton using cytochalasin D suppresses stretch-induced signaling to p70S6K1 via altering the dynamics of the actin cytoskeleton. Indeed, a direct link from the cytoskeleton to signaling activity

for loss. Although the cause(s) of sarcopenia are unknown, unlike with disuse atrophy (as will be discussed later; Section 5.4.2), aging is not associated with

gross changes in protein turnover during fasted/post-absorptive periods (i.e. depressions in synthesis or increases in breakdown).26,31,32 Rather, it has

recently come to light that aging is associated with anabolic resistance to feeding. In a nutshell this means that when exposed to equivalent amounts of AA or insulin, the capacity to increase protein synthesis and reduce protein breakdown, respectively, are diminished in elderly individuals.

The evidence for this is as follows. As previously discussed, EAA are the major drivers of muscle synthesis and the first demonstration of a decreased sensitivity and capacity for increasing muscle synthesis was made during oral feeding of EAA to men aged 65–75 years when compared to young men. For example at doses of EAA of 10 and 20 g the elderly individual’s increases in synthesis were blunted by about 50%. A possible explanation for this was reduced concentration and phosphorylation (i.e. capacity and e ciency) of the mTORc1 substrates, p70S6K1 and 4E-BP1. More recently we also identified that this blunting is not restricted to the muscle protein synthesis (MPS) arm of turnover. This is because in older individuals, the B50% inhibition of muscle breakdown in response to a modest rise in insulin availability is also blunted. As such there is anabolic resistance in both arms of protein turnover after feeding, which we propose contribute to or even cause sarcopenia. As was mentioned earlier, with aging, there are no major alterations in muscle proteostasis under post-absorptive conditions to explain sarcopenia (i.e. breakdown4synthesis). Perhaps it makes sense that small reductions in protein accretion after feeding may instead regulate sarcopenia in view of the slow, incipient wasting with which it is associated. Indeed, because protein turnover under post-absorptive conditions predominates a diurnal cycle (B19 vs. B5 h) sarcopenia should be much more rapid if di erences in post-absorptive turnover were apparent.

Significantly it was recently shown that, as for feeding, protein synthesis responses to resistance exercise are lower in older than younger men across a range of intensities when matched for work. This likely explains the observation that muscle hypertrophy is less after training in old versus young humans. These observations further add weight to the concept of anabolic blunting being key in the regulation of age-related muscle wasting.

5.4.2Disuse Atrophy

Skeletal muscles house some 40% of all protein in the body of a healthy human

but this store is depleted when habitual mechanical input (i.e. standing, walking etc.) is removed.33,34 The loss of muscle associated with muscular inactivity is

collectively known as disuse atrophy and is the product of a reduction in muscle CSA and length. Disuse atrophy occurs under many ground-based situations of reduced neural input such as whole-leg casting after fractures/ breaks, chronic bed-rest during hospitalization, denervation due to spinal cord injury, chronic sedentarism in aging populations, but also during space-flight.

As a consequence of muscle atrophy, there is loss of strength and reduced capacities of whole-body glucose storage and metabolism, which contribute to insulin resistance. Rates of muscle loss in all models of disuse are fastest within the first 30 days with a mean loss in muscle CSA of B0.6% per day.

Muscle atrophy in disuse and indeed any other wasting situation (e.g. aging, cancer, sepsis etc.) must ultimately be regulated by changes in proteostasis that favor a net loss of tissue (i.e. muscle protein synthesis is exceeded by muscle protein breakdown). The prevailing view is that reductions in synthesis, not increases in breakdown, cause disuse atrophy in humans (though the latter has not yet been measured). This is because impairments in muscle synthesis are likely su cient to explain the observed muscle loss without the need for substantial increases in muscle breakdown. For example, unlike muscle loss with aging, disuse is associated with reductions in muscle synthesis under postabsorptive conditions. Indeed, the first demonstration of this was a reported decrease of B25% in post-absorptive rates of muscle synthesis during legcast immobilization. Since then, these findings have been substantiated in other immobilization studies with the most recent study showing B50% reductions in synthesis of quadriceps muscle both 10 and 21 days after unilateral leg immobilization. Furthermore, the blunting of MPS is not restricted to postabsorptive periods. Indeed, as with aging, increases in muscle synthesis during infusion of amino acid infusions are severely blunted in immobilized human legs. Therefore, when coupled to reductions in post-absorptive muscle synthesis, this would strengthen the evidence for impairments in muscle synthesis being the key cause of human disuse atrophy. Furthermore, these findings are yet another example of anabolic blunting, adding to what was previously reported in aging and which likely contributes to atrophy in other conditions (i.e. aging, chronic obstructive pulmonary disease, cancer, type 2 diabetes etc.).

What causes blunted muscle synthesis in disuse under post-absorptive conditions or after feeding is not known. Unlike in aging, those signals typically associated with the acute upregulation of MPS after increasing AA availability to muscle (i.e. Akt/mTOR signaling) are neither suppressed under post-absorptive conditions nor blunted in fed conditions when comparisons are made between immobilized and non-immobilized legs at 14 days. Large-scale gene expression analyses (microarrays) gathered from these studies indicate that the largest downregulation of functional gene sets at both 2 and 14 days were those encoding for proteins representing all facets of mitochondrial function, including the key mitochondrial gene transcriptional co-activator peroxisome proliferator co-activator 1 (PGC1a). Since over-expression of PGC1a protects against disuse atrophy in rodents (and sarcopenia) it is plausible that sustaining mitochondrial volume prevents decreases in muscle synthesis. The second notable downregulation was in genes encoding for RNA/proteins regulating the capacity for muscle synthesis (i.e. initiation factors, ribosomal units). Thus it may be that a reduction in translational capacity rather than e ciency (i.e. phosphorylation) regulates muscle loss in response to disuse.

Whether or not increases in muscle protein breakdown contribute to atrophy in human disuse remains contentious. Calculations based upon the blunting of

synthesis over a diurnal cycle suggest that increases in breakdown are not required to explain the observed muscle loss. For example, normal turnover is 0.05% h–1 or 1.2% d–1 where synthesis and breakdown are equal and opposite. Based on data showing that muscle synthesis increases 2.5-fold after a maximally e ective feed with the increase only lasting 1.5 h, it is likely that r5 h/day is spent in ‘‘fed’’ periods where muscle is gaining protein (assuming three good meals). Thus, based on conservative assumptions from previous findings in disuse in which muscle synthesis is suppressed B50% in both post-absorptive and fed periods then diurnal protein accretion would be: (0.025 19) þ (0.025 1.25 5) ¼ 0.63% per day. If muscle protein breakdown remained constant then muscle would be lost at a rate of: 1.2 – 0.63 ¼ 0.57% per day. This is indistinguishable from the typically measured B0.6% per day over the first 30 days, which suggests that increases in muscle breakdown are not necessary to explain human disuse atrophy. Nevertheless, some work has provided some secondary evidence from static markers (e.g. increased expression of proteasomal subunits) for short-term increases in degradation. For example, increases in interstitial 3-methylhistidine (a marker of myofibrillar breakdown) was reported to occur 3 days after immobilization as was upregulation of total ubiquitinylation and mRNA for the E3 ubiquitin ligases and so-called ‘‘atrogenes’’, muscle ring-finger 1 (MuRF1) and muscle atrophy F box (MAFBx), which have been suggested as a common mechanism for muscle wasting (see ref. 35 for further detail). Additionally, other workers have shown that during short-term immobilization, for 5 days and after spinal cord transection in patients, there are increased amounts of markers of increased protein breakdown, in terms of both mRNA and protein. Furthermore, in animals, there is also evidence that acute disuse may cause damage to mitochondria, leading to the generation of ROS and oxidative stress, which themselves have been purported to increase apoptotic and proteolytic processes in muscle. One possibility that has been overlooked is that such increases in markers of breakdown are not necessarily regulating ‘‘bulk’’ increases in protein breakdown, but rather targeted degradation. For example, MAFbx targets the eukaryotic initiation factor 3 subunit 5 (eIF3-f) for ubiquitination and degradation by the proteasome and since mTOR and p70S6K1 interact directly with eIF3-f to mediate assembly of the translation pre-initiation complex this could be the explanation for reduced muscle synthesis, rather than proposed increases in protein breakdown.

5.4.3Sepsis

Sepsis due to infection induces whole body inflammation and is a major cause of comorbidity and mortality in critically ill patients and is associated with and exacerbates acute trauma and thermal injury.36,37 Wasting of body muscles is associated with sepsis and this can directly contribute to the morbidity and mortality, particularly when the respiratory muscles are involved. While sepsis has negative consequences and is associated with illness, the induction of the inflammatory state is presumably adaptive in as much as liberation of energy

from various tissues can assist in healing. As such, there is a large induction of glucocorticoids, proinflammatory cytokines and increases in cellular Ca21 in sepsis. Collectively, this results in increased protein degradation. This increase is predominantly via proteasome-mediated degradation and is associated with upregulation of MURF-1 and MAFbx mRNA, likely due to a downregulation in PI3K/Akt signaling. However, both the calpains and caspases are activated and thought to initiate breakdown of myofibers with final degradation occurring via the proteasome. As cytokines all promote degradation via the proteasome, these factors are presumably extramuscular signals responsible for triggering muscle degradation in septic patients. However, decreased protein synthesis and mitochondrial dysfunction have been observed in septic patients. Presumably the decreased protein synthesis could be the result of elevated cortisol, TNFa and/or Ca21 in septic patients as a result of degradation of key pieces of the transcriptional and translational machinery (for example transcription factors, initiation and/or elongation factors). Indeed, both glucocorticoid and Ca21 antagonists (RU38486 and dantrolene, respectively) have e cacy in reducing muscle wasting in experimental sepsis. Similarly, mitochondrial dysfunction and altered plasma membrane conductance could be due to increased membrane and/or protein damage arising from the increased ROS being present in inflamed tissue as the result of release from inflammatory cells (for example macrophages and neutrophils).

5.4.4Burns

Burn patients often enter a hypermetabolic state where energy is used in healing the wound and raising core body temperature.38 Typically this hypermetabolic state is observed for burns covering 10% or more of the total body surface area and there is a proportional relationship between size of the burn and the resting metabolic rate. When left untreated the muscle wasting associated with burn contributes to both comorbidity, for example infection, and mortality, particularly in patients with larger burn surface areas. Successful treatment modalities point to the complexity of the muscle wasting observed.

Muscle wasting appears to occur via at least four distinct mechanisms. Firstly, in the immediate post-burn period there is a hypercatabolic state, which includes increased muscle protein degradation. The initial hypercatabolic state can be eased by excision and closure of the burn, for example a 40% reduction for large burn surface areas that are excised and covered after two to three days versus after one week. This suggests that at least part of the catabolic state is directly tied to the increased requirement for heat production. While muscle is the major metabolically active tissue in the body and therefore the major producer of heat due to ine ciency of running oxidative phosphorylation, the notion that human muscle functions to produce heat is controversial and therefore the mechanisms controlling increased heat production are currently largely unknown. Presumably increased mitochondrial uncoupling occurs in muscle via increased expression of uncoupling proteins. Thyroid hormone and epinephrine which, as stress-induced hormones, are likely both elevated