During limb immobilization, the rate of protein synthesis and degradation is affected by the removal of a mechanical stimulus [30], thereby triggering the ubiquitin proteolytic system (UPS), calcium-activated protease system (CAS), and myostatin to carry out skeletal muscle proteolysis. Protein degradation via the UPS is carried out by the ubiquitin molecule and a series of enzymes (E1, E2, E3) tagging a specific protein for destruction. The protein is then taken into the 26S proteasome where degradation occurs [31, 32]. Calpains mediate skeletal muscle degradation through digestion of individual myofibrillar proteins [33–37] and tend to influence gene expression through the cleavage of specific transcription factors, thereby affecting the UPS [38]. Research regarding myostatin suggests that expression may be associated with muscle myosin heavy chain isoform IIb [25], as Willoughby et al. [22] noted a 125% increased expression of myostatin mRNA in the immobilized human gastrocnemius, which is composed of approximately 76% of type II muscle fibers [39].

Currently, there is no evidence supporting the involvement of tumor necrosis factor-α (TNF-α) in disuse atrophy [5]. However, administration of TNF-α has been shown to activate cachexia-induced protein degradation [40, 41] and increase ubiquitin mRNA expression and activity of ubiquitin conjugates in skeletal muscle. TNF-α also activates nuclear factor-κB (NF-κB), which is involved in immune and inflammatory responses [42], and has been shown to increase myofibrillar proteolysis and suppress myosin synthesis via the UPS [12, 40, 43–45]. Together, TNF-α and NF-κB upregulate E2 and the 20S proteasome in the UPS during diseased states [5, 41, 46, 47]; however, current research shows that TNF-α is unchanged with disuse-induced atrophy in humans even though NF-κB levels are markedly increased [12, 42, 48]. This suggests that disuse-induced atrophy may activate NF-κB through an alternative pathway in humans.

In conjunction with previous research, results also indicated that 28 days of immobilization resulted in significant increases in the mRNA expression for the ubiquitin protein, E2, E3, 20S-HC2, 20S-HC3, atrogin-1, and MuRF1 [1, 8, 22, 67, 68]. No other study has shown an increase in the mRNA expression of E3, but this change may indicate a greater need for polyubiquitination of muscle proteins and thus allow for greater protein degeneration. Overall, the results indicate that there appears to be a comprehensive increase throughout the entire UPS pathway. Additionally, the increased expression of calpain 1 and calpain 2 agrees with previous studies indicating that calpains affect muscle protein degradation [33, 35, 36]. This finding indicates that calpains play a role not only in protein degradation in diseased states [69] and during damaging exercise performance [70], but also during disuse atrophy.

No previous study has shown evidence that TNF-α is involved in disuse atrophy in humans [5]; instead, it has been shown to activate NF-κB in several diseased states resulting in muscle atrophy [14, 41, 71, 72]. Researchers speculated that NF-κB worked independently from TNF-α during immobilization in humans because evidence showed no change in TNF-α during unloading, yet still resulted in an increase in NF-κB [12, 42, 48] and atrophy. However, data from the current study indicate that both TNF-α and NF-κB mRNA levels were increased. It should be noted that with these data an alternative pathway may not be necessary to activate the degenerative effects of NF-κB, but there may also have been an increase in transcription of these markers without an increase in translation, or perhaps an increase in these markers, without activation. Therefore, more human research is necessary in order to observe this potential association between the UPS and TNF-α and NF-κB in order to further elucidate the route by which the cytokines function as signaling mediators of muscle protein degradation.

In agreement with Reardon et al. [26], increases in mRNA expression of myostatin seen in the present study indicate a strong influence of myostatin on the inhibition of muscle growth as a result of immobilization. However, these results are conflicting with Jones et al. [1] and McMahon et al. [73] who did not observe any changes in myostatin during hind-limb unloading. Myostatin may be a fiber type-specific inhibitor of muscle growth, suggesting a stronger association between myostatin and type IIb muscle fibers [25]. The gastrocnemius is composed of about 50% slow twitch fibers and 50% fast twitch fibers [74], indicating a greater amount of fast twitch fibers than that of the soleus and digitorum longus studied by McMahon et al. [73]. However, Jones et al. [1] showed no changes in myostatin in the vastus lateralis, which is composed of approximately 68% type II fibers [74]. Overall, the conflicting evidence of myostatin's role within muscle atrophy during immobilization renders the need for more research in order to show specific effects and action on the various muscle fiber types.

The results of this study showed that the PAA supplement ingested was not influential in attenuating the expression of myostatin and the ubiquitin and calpain proteolytic pathways. PAA supplementation has been effective in maintaining a higher protein fractional synthesis rate during disease-induced and unloading-induced proteolysis [4, 59, 60, 75] and following damaging eccentric exercise by stimulating muscle protein synthesis, but showed little to no effect on muscle protein degradation [49, 51]. Results from the current study could be different from existing literature because ingestion of extra dietary PAA may not effectively alter the physiological changes in the body that are induced by muscle unloading. It is suggested that amino acid catabolism and whole body protein turnover is upregulated during unloading and immobilization [2], and in as little as 14 days of bed rest whole body protein turnover decreases by 15%, with 50% of the decrease coming from an attenuation of protein synthesis [29]. Research also indicates that during unloading and disuse conditions, the decrease in protein synthesis appears to drive the loss of muscle mass, while the rate of protein degradation remains fairly constant [28, 29]. While there appeared to be no effect on net protein turnover as observed through maintenance of leg lean mass, this may indicate a potential benefit of PAA or PLA on protein synthesis. Previous research [76] has shown that ingesting 1.5g of protein per kg of body weight per day was the optimal protein intake in the prevention of sarcopenia in the elderly. The participants in the PAA group exceeded this amount with their dietary intake and ingestion of the PAA supplement (Day 14 mean intake 2.2 ± 0.7g/kg/d; Day 28 mean intake 2.2 ± 0.6g/kg/d). This information suggests that the optimal protein intake for younger males aged 18–30 years old may need more daily protein than the elderly to prevent muscle loss during immobilization. Thus, the amount of dietary protein ingested may not have been enough to attenuate protein synthesis and effectively alter proteolytic pathways during immobilization.