Research articleSixty days of head-down tilt bed rest with or without artificial gravity do not affect the neuromuscular secretome
Introduction
Artificial gravity generated by human centrifugation is a potential countermeasure to mitigate the effects of weightlessness during long-term space missions [[1], [2], [3], [4]]. Human centrifugation can be applied in a continuous or intermittent fashion. The efficacy of both alternatives has not yet been directly compared in a long-term bed-rest study. In a 5-day bed-rest study, intermittent centrifugation with 1G at the centre of mass was better tolerated than continuous centrifugation and was associated with lower adrenocortical stress responses [5] and attenuated reductions in orthostatic intolerance [6] compared to continuous centrifugation. In contrast to these positive effects of human centrifugation, neither continuous nor intermittent centrifugation counteracted bed-rest-induced changes in cardiac function [7] and the catabolic effects on muscle and bone metabolism [8]. However, in a 21-day bed-rest study, daily exposure to 1 h of continuous artificial gravity with 2.5 G at the feet, similar to 1 G at the centre of mass, had a small attenuating effect on losses in muscle mass and function, possibly mediated by a blunted increase in muscle myostatin content [9] and preserved rates of muscle protein synthesis [10]. It remains to be seen whether the attenuated bed-rest-induced atrophy after 21 days, but not after 5 days, is related to the duration and hence more accentuated after a long period of bed rest.
Deconditioning of the neuromuscular system, characterized by muscle weakness, is one of the major physiological responses to microgravity during long-term space missions and in bed rest [[11], [12], [13], [14], [15], [16], [63], [65]]. While muscle weakness during disuse is largely attributable to muscle atrophy, the contribution of an impaired motor drive may be an important factor for the loss of muscle strength, particularly during the early stages of disuse. Such impaired activation capacity may be the consequence of disuse-induced remodelling of the neuromuscular junctions (NMJ) [17] as an adaptation to the reduced number of nerve impulses reaching the motor end plate [18]. This adaptation of the NMJ can impair postsynaptic signal transduction and cause defects in the excitation-contraction coupling [66]. The affected post-synaptic signalling can in turn activate apoptosis and proteolysis, and impair the regenerative potential of the muscle [[19], [20], [21]]. Neurotrophins and neurotrophic factors play an important role in maintaining the integrity of the NMJ [18,22,23]. Disuse-induced problems with neurotransmission and synaptic vesicle recycling may well be a consequence of a reduction in the levels of these circulating factors. It has been found that the circulating [24,25] and muscle [[26], [27], [28]] expression levels of some neurotrophic factors correlate positively with the extent of muscle loading. For example, in muscle the expression of glial cell line-derived neurotrophic factor (GDNF), important for maintenance of the NMJ [26,27], was markedly decreased with hind limb suspension and increased with exercise [28]. Daily treadmill exercise increased serum levels of GDNF and Brain Derived Neurotrophic Factor (BDNF) in rats undergoing peripheral nerve repair after nerve transection [25]. In line with these rodent studies, the bed-rest-induced increase in BDNF in old, but not young people, was suggested to be a neuroprotective response [29,30]. In addition to neuroprotection, the concomitant increase in circulating BDNF after muscle-damaging exercise and satellite cell activation [31] suggests that BDNF also facilitates muscle recovery from damage. Although no significant change in BDNF has been observed in young participants after 14 days of bed rest [29,30], it is possible that bed-rest-induced decrements in BDNF occur later in younger than older people and can be prevented by human centrifugation.
C-terminal Agrin Fragment (CAF) blood levels are suggested to be a biomarker for NMJ fragmentation and are used as a clinical marker for the progression of sarcopenia [[32], [33], [34]]. Elevated levels of the synaptic protease neurotrypsin cause a loss of NMJ integrity by fragmentation of agrin into the soluble 22 kDa C-terminal Agrin Fragment that can lead to muscle wasting [35,36]. CAF levels have not been previously reported for bed rest or spaceflight, but are expected to rise in case of NMJ fragmentation during spaceflight and bed rest.
With regards to markers of muscle wasting, Creatine kinase and slow skeletal muscle troponin T are well-known makers and have been reported to be elevated in immobilization [37]. In addition, urine levels of prostaglandin E2 were decreased during Space Shuttle flights [38] and muscle myostatin levels were increased after 84 days of bed rest [39]. In a recent study on mice that spent 33 days on the International Space Station, it was shown that systemic inhibition of myostatin/activin A signalling led to increases in both muscle and bone mass, with comparable effects in ground and flight mice [40]. The myostatin signalling pathway is thus a potential target for the development of medications to serve as a countermeasure to muscle and bone loss. While the muscle levels of myostatin were elevated during bed rest, this was prevented by concomitant flywheel exercise [39], but nothing is known about the circulating levels of myostatin and the potential impact of human centrifugation.
To gain insight into the possible factors that affect the neuromuscular integrity during human centrifugation in a long-term bed-rest study we measured the time course of bed-rest-induced changes in neurotrophic factors, markers of muscle wasting and NMJ fragmentation. We hypothesized that bed rest is accompanied by transient changes in the levels of circulating neurotrophic factors, circulating markers of muscle wasting and C-terminal Agrin Fragment that are attenuated by intermittent and continuous artificial gravity.
Section snippets
Ethical approval
The study was approved by the IRB of North Rhine Medical Association (Ärztekammer Nordrhein, application number 2018143) in Düsseldorf, Germany, the IRB of Hospital Maastricht, The Netherlands (application number NL68345.068.18/METC 19–001), and by the IRB of NASA's Johnson Space Center, Houston, Texas, USA. It was registered in the German Clinical Trials Register (DRKS-ID: DRKS00015677).
Experimental design
The Artificial Gravity Bed Rest – European Space Agency (AGBRESA) study was a joint project of the European
Results
All blood samples were successfully obtained and processed as planned. At baseline, there was no significant difference between groups (Table 1). The analyses revealed no significant differences between men and women, and no significant effects of bed rest or the interventions on any of the measured parameters. In detail, the following findings were obtained.
Discussion
The aim of the present study was to assess 1) the immobilization effects of bed rest on the neuromuscular interaction secretome and 2) whether any immobilization-induced changes in the secretome could be attenuated by daily continuous or intermittent human centrifugation with 1 G at the centre of mass. The main finding was that none of the measured parameters including serum markers of muscle wasting (GDF-8/myostatin, slow skeletal muscle troponin T, creatine kinase, prostaglandin E2),
Conclusions
Our findings indicate that long-term bed rest or artificial gravity with 1 G at the centre of mass for 30 min per day had no effects on the neuromuscular secretome. Artificial gravity neither resulted in changes in neurotrophic factors such as BDNF, GDNF or CAF, nor in circulating markers of muscle wasting, indicating that it is a safe intervention that does not have negative effects on neuromuscular integrity.
Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Role of the funding source
The authors acknowledge the German Research Foundation (DFG, grant number GA 2420/1-1), German Aerospace Center (DLR, grant number 50WB1928) and the UK Space Agency (ST/S0001735/1 & ST/T00066X/1) in funding this project. In addition, the authors appreciate the support of the European Space Agency (ESA, 16-16ESA AGBR-0013, contract number 4000113871/15/NL/PG) and the National Aeronautics and Space Administration (NASA, contract number 80JSC018P0078). The DFG provided a research fellowship grant
Credit author statement
Bergita Ganse, Conceptualization, Investigation, Data curation, Visualization, Writing – original draft preparation. Alessandra Bosutti, Methodology, Writing-reviewing and editing. Michael Drey, Funding acquisition, Supervision, Writing-reviewing and editing. Hans Degens, Resources, Supervision, Formal analysis, Writing-reviewing and editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
Special thanks for the great operational support of Dr. Jennifer Ngo-Anh (ESA), Dr. Edwin Mulder, Dr. Jessica Lee and the entire team of the DLR Institute of Aerospace Medicine in Cologne during the study.
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