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Figure 5.2 Extracellular/intracellular signaling in the regulation of muscle proteostasis by muscular activity and nutrients.

may be in part because some signaling proteins are physically immobilized to the cytoskeleton and are thus sensitive to alterations in cytoskeletal dynamics. In addition, inhibition of contraction induced phospholipase D (PLD1) activity reduces production of the lipid second messenger, phopsphatidic acid (PA) thereby attenuating signaling to p70S6K1 and muscle protein synthesis. On the other hand, inhibiting ATPase activity during contraction with N-benzyl-p- toluenesulfonamide suppresses Ca21-mediated increases in eEF2 phosphorylation (which normally acts to suppress protein synthesis) and in doing so attenuates the blunting of protein synthesis normally seen during muscle contraction. Also, increases in cellular AMP : ATP ratios during contraction promotes activation of AMPK, which can have a negative influence on protein synthesis. Unfortunately, although the potential exists for the influence of other factors generated during contraction such as ROS and other purinergic signals (UTP, ADP etc.) to modulate muscle proteostasis this remains to be functionally confirmed. Nevertheless, once again these findings again highlight the close relationship between energy and protein metabolism.

5.4Conditions Associated with Alterations in Muscle Proteostasis in Humans

Amino acids are released from muscle protein during wasting conditions such as aging, starvation, sepsis, chronic obstructive pulmonary disease (COPD), thermal

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Table 5.1 Alterations in proteostasis under conditions of muscle wasting in humans.

	Postabsorptive	Postabsorptive	Anabolic response to
Condition	synthesis	breakdown	feeding
Starvation	k	k (m)?	?
Trauma/sepsis	k or m	m	?
Burns	k	m	?
Cancer cachexia	k	k(m)?	k
Muscular dystrophy	k	k	?
Sarcopenia	¼	¼	k
Resistance exercise	m	¼or m	?

injury (burns), acute trauma, HIV/AIDS, diabetes, renal failure and some aggressive forms of cancer (i.e. pancreatic). Under these circumstances muscle wasting may be extremely rapid i.e. noticeable within days. On the other hand, loss of muscle protein with aging as discussed below is a slow, incipient process. Depending on the catabolic insult and its severity, loss of muscle mass results from decreased, normal or even increased protein synthesis, which in the latter case remains insu cient to compensate for higher proteolysis. Changes in proteostasis that occur in a variety of muscle wasting conditions are illustrated in Table 5.1.

Is there a physiological foundation for muscle breakdown under pathological circumstances? In short the answer is yes, because release of amino acids from muscle provides substrate for hepatic gluconeogenesis, supports acute phase protein synthesis and the immune system (i.e. high rate of glutamine utilization in lymphocytes, macrophages and neutrophils), and is an important energy source to enterocytes. As such, at least in the short term, this is an adaptive response that is beneficial to support the function of certain vital tissues, for example during the acute phase of sepsis.

However, during situations of sustained muscle wasting such as severe protracted sepsis, cancer cachexia or burn injury, the benefits of muscle loss will be outweighed by the costs. This is for several reasons. Firstly, as a consequence of muscle weakness, ambulation is delayed and as such this increases thromboembolic episodes. Moreover, forced inactivity due to muscle weakness (i.e. disuse atrophy) further exacerbates muscle wasting by superimposing onto the initial cause of muscle loss. Muscle wasting of respiratory muscles also increases reliance upon ventilatory support, which leads to increased weaning di culties and, consequently, morbidity and mortality. Therefore, muscle wasting exacerbates clinical outcome. In fact, loss of B30% of muscle mass results in death. An extensive discussion of the regulation of atrophy in all situations of muscle loss is not possible due to the large numbers of pathological situations associated with muscle wasting; as such only a select number are discussed.

5.4.1E ects of Aging on Muscle Proteostasis

Aging is accompanied by a loss of skeletal muscle mass, which is defined as sarcopenia, a word adopted from Greek roots i.e. ‘‘sarx’’ for flesh and ‘‘penia’’

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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.

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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