A glimpse into the early window of muscle unloading

Maintaining skeletal muscle mass is essential for normal ambulatory function and metabolism. The rapid onset of muscle loss (atrophy) resulting from prolonged disuse (e.g. bedrest, casting or sedentary lifestyle) is a significant obstacle towards preventing chronic disease and maintaining functional independence. Coinciding with atrophy, there is an unfavourable and disproportionate loss of strength and power (dynapenia) compared to loss of mass as a result of prolonged disuse (Arentson-Lantz et al. 2016). Such disparate declines in muscle strength have been observed after only 3 days of restricted mobility via dry immersion (Demangel et al. 2017). Although dry immersion is an extreme form of disuse, these data underscore the need to understand the mechanism(s) by which contractile function is more negatively impacted by disuse. However, the molecular and cellular factors driving such drastic declines in strength compared to mass from unloading have yet to be fully elucidated. In a recent study issue of The Journal of Physiology, Monti et al. (2021) may provide an answer that explains why declines in strength are greater than loss in mass following prolonged periods of disuse. To induce skeletal muscle atrophy Monti et al. (2021) subjected 10 relatively homogeneous healthy males to bedrest for 10 days (BR10) under 24 h surveillance. Reduced muscle volume and cross-sectional area of the vastus lateralis and quadriceps femoris muscles were confirmed via magnetic resonance imaging and ultrasound, respectively. Strength loss significantly exceeded the percent decline seen in muscle mass as evidenced by reduced maximal voluntary contraction and explosiveness (time-to-63%-force) with knee dynamometry. However, activation capacity measured through the interpolated twitch technique remained unchanged after BR10, suggesting that myofibrillar motor recruitment remained intact and therefore was probably not a contributor to lower force production. Similar declines in strength have been observed following 3 days of dry immersion but were also accompanied by signs of partial fibre denervation, implicating possible neuromuscular junction (NMJ) involvement (Demangel et al. 2017). The NMJ plays an instrumental role in the motor neuron-muscle fibre recruitment axis as the site of chemical transmission between the neuron and muscle fibres, initiating contraction. However, the loss of synapses (denervation) can result in marked declines in muscle recruitment and strength. Monti et al. (2021) thus hypothesized that a disruption to the NMJ may underly the disparity in force loss compared to muscle size loss during disuse. Interestingly, Monti et al. (2021) found redistributed, cytosolic staining of neural cell adhesion molecule (NCAM), indicative of denervation. NCAM redistribution along the sarcolemma after BR10 coincided with increased C-terminus agrin fragment (CAF) in plasma, a marker associated with neuromuscular damage and commonly observed in age-related muscle loss (sarcopenia). These data, together with altered expression of genes related to NMJ (e.g. AGRN, CHRNA1 and HOMER2), suggest that just 10 days of bedrest may be sufficient to result in a loss of NMJ stability. However, a recent study demonstrated that CAF does not change following 60 days of bedrest in younger people (Ganse et al. 2021). This may point to methodological differences between these studies suggesting that possible confounding variables may impact CAF levels during bedrest and necessitate future investigation. To gain insight into how impaired NMJ stability may underlie muscle function loss after 10 days of bedrest, Monti et al. (2021) developed a single-fibre method for assessing excitation-contraction coupling function ex vivo. Their method utilized calcium (Ca2+) and caffeine to induce single fibre contraction allowing a maximal release of calcium from the sarcoplasmic reticulum (SR), thus permitting assessment of calcium dynamics. At BR10, no change in specific force was detected in response to exposing the fibre to a high Ca2+ concentration (pCa = 4.5) indicative of normal contractile motor function. With this procedure, one can directly test the functioning of the myofibrillar machinery bypassing the SR and Ca2+ release. Contractile response to caffeine-induced calcium release was impaired at BR10, as seen by the significant reduction of calcium concentrations in response to a maximal contraction following a prolonged SR calcium loading period. Furthermore, to determine possible deficiency in the uptake and release of calcium, the function of the SR was tested by again inducing a maximal contraction, although this time without a calcium loading phase, thus determining the residual content of calcium in the SR resulting in significant reductions after BR10. These findings suggest that periods of disuse result in either incomplete release of calcium from the SR or incomplete sequestering after BR10. This novel observation of impaired calcium handling following bed rest may implicate SR dysfunction as a contributing factor to the noted NMJ disruption and associated loss of strength. In support of impaired calcium release following 10 days of bed rest, Monti et al. (2021) investigated several molecular targets involved in calcium release, storage and uptake. By BR10, sarcoendoplasmic reticulum calcium transport ATPase isoform 2 (predominantly expressed in type 1 fibres), for which the role is to return calcium to the sarcoplasmic reticulum, was reduced by 50%. Similarly, the expression for calsequestrin 2 (a key determinant of calcium storage notably absent in type II muscle fibres), which buffers calcium ions to coordinate contraction and relaxation, was dramatically reduced by BR5 and BR10. These findings, in conjunction with an increase in MYH1 and potential decrease in MYH7 gene expression, implicate a slow-to-fast phenotypic switch. Furthermore, the gene encoding for ryanodine receptor 1 was more than doubled by BR10. The ryanodine receptor mediates release of calcium into the cytosol, which may be a compensatory response analogous to the increased compound muscle action potential that occurs with ageing in an effort to maintain strength with atrophy (Urso et al. 2006). Altogether, bedrest appears to disrupt calcium handling in the sarcoplasmic reticulum. This occurs especially, but not exclusively, in type 1 fibres by down-regulating important sequesters and pumps necessary for muscle contraction. As alluded to by Monti et al. (2021), a noted decline in mitochondria could potentially contribute to the observed NMJ dysfunctions following prolonged bedrest. Mitochondria are found in abundance around the synaptic cleft and motor neuron to provide energy for synaptic transmission (Rygiel et al. 2016). Mitochondrial energetic production is partially influenced by intracellular calcium concentrations. Future experiments should clarify the role of mitochondria and reactive oxygen species in bedrest-induced NMJ instability and calcium mishandling given the documented aberrance of each in response to disuse and sedentarism, with the latter leading to increased oxidative stress, known also to occur in ageing skeletal muscle. Monti et al. (2021) observed a novel trend toward decreased TOM20 protein expression and significant reductions in PPARGC1A mRNA, suggesting decreased mitochondrial content and reduced mitochondrial biogenesis, respectively. Together, this supports a role for mitochondrial dysfunction in disuse-associated maladaptations to the NMJ and could be a consequence of impaired SR-mediated calcium release. NMJ dysfunction and mitochondrial aberrations are implicated in sarcopenia, creating a possible link between disuse induced NMJ instability and mitochondrial dysfunction. As alluded to by Monti et al. (2021), how these elements influence and are influenced by trophic factors released by motor neurons (such as brain-derivedneurotrophic factor, insulin-like growth factor-1) is worthy of future research aiming to mitigate bedrest-induced NMJ dysfunction as a result of their potential to enhance synaptic stability. The current work of Monti et al. (2021) provides novel understanding of the molecular underpinnings of the functional declines seen during prolonged bedrest that may improve detection and subsequent mitigation of the functional declines caused by disuse. Moving forward, the disuse field will undoubtedly interrogate the dynamic NMJ as an initial site of skeletal muscle atrophy. As a functional measure, Monti et al. (2021) used knee dynamometry. However, examining negative changes in performance with coordination- and balance-intensive functional movements consisting of universal motor recruitment patterns and multiple joints (such as standing up from seated, i.e.‘squatting’, as well as gait patterns) would be important areas for further study because these measures could capture maladaptive changes within skeletal muscle that better translate to impaired activities of daily living. Furthermore, such findings could also redirect attention to the neural adaptations of resistance exercise to improve physical therapy outcomes for atrophy-affected individuals. Monti et al. (2021) note that, although peripheral and central factors are probably at play, their study is importantly the first to highlight what may be the initial event (NMJ disruption) in muscle impairment following disuse. The work conducted by Monti et al. (2021) beckons the field of muscle plasticity to examine the NMJ itself with respect to further expanding the knowledge of disuse physiology. Eventually, this research may catalyse the innovation of potent interventions for anyone suffering from immobilization as a result of disease or age-related declines such as sarcopenia and dynapenia. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. The authors declare that they have no competing interests. Both authors equally wrote, edited and approved the final version of the manuscript submitted for publication. Matt Brisendine is funded by a graduate research assistantship through the Human Nutrition Foods and Exercise Department at Virginia Tech. Jacob Bond is funded by a graduate research assistantship through the Human Nutrition Foods and Exercise Department at Virginia Tech and the Translational, Biology, Medicine, and Health Program at Virginia Tech. We thank our mentors Dr Siobhan Craige and Dr Josh Drake for their guidance, feedback and patience.