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Systemic redox biomarkers suggest non-redox mediated processes in the prevention of bed rest-induced muscle atrophy after exercise training: The Cologne RSL study
Acta Astronautica 168 (2020) 116–122
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Research paper
Systemic redox biomarkers suggest non-redox mediated processes in the prevention of bed rest-induced muscle atrophy after exercise training: The Cologne RSL study
T
C.F. Dolopikoua, I.A. Kourtzidisa, A.N. Tsiftsisa,b, N.V. Margaritelisa,b, A.A. Theodorouc, V. Paschalisc,d, C.A. Frantzidise,f, M.G. Nikolaidisa, C. Kourtidou-Papadelif,g, A. Kyparosa,f,∗ a
Department of Physical Education and Sports Science at Serres, Aristotle University of Thessaloniki, 62110, Serres, Greece Intensive Care Unit, 424 General Military Hospital of Thessaloniki, 56429, Thessaloniki, Greece Department of Life Sciences, School of Sciences, European University Cyprus, 1516, Nicosia, Cyprus d School of Physical Education and Sport Science, National and Kapodistrian University of Athens, 17237, Athens, Greece e Laboratory of Medical Physics, Medical School, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece f Greek AeroSpace Medical Association – Space Research (GASMA-SR), 44 Ethnikis Antistasis, 55133, Thessaloniki, Greece g Aeromedical Center of Thessaloniki (AeMC), 44 Ethnikis Antistasis, 55133, Thessaloniki, Greece b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Atrophy Eccentric exercise Muscle Oxidative stress Redox Blood Urine
It has been previously reported that eccentric-biased exercise training prevents the decreases in lean body mass after 60 days of head tilt down bed rest (“Cologne RSL Study”). The aim of the present study, as a part of Cologne RSL Study, was to investigate whether these anti-atrophy effects of exercise training are regulated by redox processes, as assessed indirectly via redox biomarkers in blood and urine. Twenty-four volunteers (N = 24) participated in a randomized controlled study and were randomly divided into two groups: a jump training group (JUMP, n = 12) that performed a specific eccentric-biased training protocol on a Sledge Jump System and a control group (CON, n = 11; one drop-out) that did not perform any exercise. All participants maintained a 6° head tilt down position for 24 h/day for 60 days. Redox measurements in plasma, erythrocytes and urine were performed at several time points throughout the study (i.e., baseline, intervention and recovery phases). A main effect of time was found for all dependent variables (P < .05). In particular, plasma protein carbonyls, erythrocyte catalase activity and urine F2-isoprostanes increased, while erythrocyte glutathione concentration decreased over time in both groups. In contrast, neither a main effect of group nor a significant group × time interaction was found in any of the measured variables (P > .05). In conclusion, our findings in systemic redox biomarkers indicate that the anti-atrophy effects of exercise training during a 60-day bed rest protocol are not regulated by redox processes.
1. Introduction Microgravity is a unique extraterrestrial component of the spaceflight environment, along with cosmic radiation, that severely challenges human health by triggering complex molecular and biochemical redox cascades [1]. Diverse spaceflight and Earth-based analog studies have shown that redox homeostasis is severely disturbed in cells and tissues. In particular, increased oxidative stress levels have been reported during spaceflight as well as in experimental set-ups that aimed to simulated space environments in vitro, ex vivo or in situ, including
various cells and tissues: rat and mouse blood after gamma rays exposure or flight at the International Space Station [2,3], rat skeletal muscle after tail-suspension or hindlimb unloading [4,5], human umbilical vein endothelial cells subjected to flight [6], rat neuronal cells after being seeded onto a high aspect rotating-wall vessel [7], mouse retina [8] and skin [9] after flight at the Space Shuttle Atlantis (STS135), rat liver after flight at STS-63 [10] and mouse brain after hindlimb suspension [11]. Interestingly, it has been demonstrated that the time course of oxidative stress varies during spaceflight, with the return to Earth acting as a late-stage oxidant stimulus [12]. The aberrant redox
∗
Corresponding author. Department of Physical Education and Sports Science at Serres, Aristotle University of Thessaloniki, Agios Ioannis, 62110, Serres, Greece. E-mail addresses: [email protected] (C.F. Dolopikou), [email protected] (I.A. Kourtzidis), [email protected] (A.N. Tsiftsis), [email protected] (N.V. Margaritelis), [email protected] (A.A. Theodorou), [email protected] (V. Paschalis), [email protected] (C.A. Frantzidis), [email protected] (M.G. Nikolaidis), [email protected] (C. Kourtidou-Papadeli), [email protected] (A. Kyparos). https://doi.org/10.1016/j.actaastro.2019.12.002 Received 6 October 2019; Received in revised form 29 November 2019; Accepted 2 December 2019 Available online 05 December 2019 0094-5765/ © 2019 IAA. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. The study design of the Cologne RSL Study. BDC: baseline data collection; HDT: head-down tilt position; R: recovery phase.
2. Materials and methods
homeostasis in response to microgravity is also exemplified by the decreased expression and/or activity of major antioxidant enzymes [13–15], the increased ROS production in cell lines [7,16,17], the impaired cellular response to a redox stimulus [18] and the decreased levels of non-enzymatic antioxidants in blood and tissues [11,19–21]. Microgravity also conveys many challenges to the human physiology affecting almost all organs and systems. Taking into account that spaceflights frequently last up to few months, these challenges have to be addressed. Muscle atrophy and the associated loss of muscle force and function have been recognized as a major threat during spaceflight [22,23]. Despite the incredible physical fitness of astronauts, muscle atrophy is one of the earliest complications observed under microgravity. From a redox perspective, it has been hypothesized that the pro-oxidative environment of skeletal muscle exacerbates atrophy via massive oxidation of contractile proteins leading to the activation of the proteolytic mechanisms [24], while nitrosative stress has also been reported to contribute via S-nitrosylation of contractile and other functional proteins in atrophic muscles [25]. Nevertheless, whether oxidative stress is the cause, the consequence or just an epiphenomenon of skeletal muscle atrophy, especially during spaceflight, remains still elusive [26,27]. Bed rest in a head-down tilt position and the hind-limb unloading method represent the two most frequently used models to simulate microgravity conditions. Both conditions result to a cephalad fluid shift and a severe decline in mechanical loading of skeletal muscles, two fundamental characteristics of microgravity. However, the bed rest model is most frequently applied in studies with human subjects compared to the hindlimb unloading model which is typically used in studies with rodents [28,29]. There are several studies that used the bed rest model for prolonged periods equal to 90 [30], 60 [31,32], 55 [33], 35 [34] and 14 days [35]. There are also some papers indicating that only a week [36] or even 5 days [37] of bed rest are enough to facilitate protein degradation and hence lead to muscle atrophy. Recently published data of the “Cologne RSL Study” demonstrated that the eccentric-biased exercise training prevented the decrease in lean body mass and lean leg mass elicited during a 60-d bed rest protocol [38,39]. Exercise as a redox stimulus transiently increases free radical production, yet in the long term it upregulates antioxidant mechanisms and decreases resting oxidative stress levels [40]. Taking also into account that exercise-induced oxidative stress is an essential signal for adaptations [41], it would be interesting to know if the effect of exercise as a countermeasure against the bed rest-induced muscle atrophy is redox-mediated. Thus, the aim of the present study, as part of the Cologne RSL Study, was to investigate whether the anti-atrophy effects (reported in Refs. [38,39]) of eccentric-biased exercise training during 60 days of 6° head tilt down bed rest are mediated by redox processes, as assessed indirectly via redox biomarkers in two different body fluids (i.e., blood and urine).
2.1. Participants Twenty-four male non-smoker adults aged 20–45 years old, with a body mass index between 20 and 26 kg/m2 were selected among 494 original volunteers based on specific physiological, psychological and medical inclusion criteria, described in detail by Kramer et al. [38]. All participants gave written informed consent to participate in the experimental procedures, which were approved by the Ethics Committee of the Northern Rhine Medical Association (Ärztekammer Nordrhein) in Düsseldorf, Germany, and the Federal Office for Radiation Protection (Bundesamt für Strahlenschutz). Participants received a financial reward for participating in the study.
2.2. Study design The volunteers (N = 24) participated in a randomized controlled training study, conducted at the Envihab, a medical research facility of the Institute of Aerospace Medicine at the German Aerospace Center (DLR) in Cologne, Germany. The study was accomplished in 2 campaigns, the first one taking place in August 2015 and the second one in January 2016. The entire study design is illustrated in Fig. 1. Each campaign lasted 90 days; 15 days of baseline data collection (BDC-15 through BDC-1), followed by a 60-d intervention phase (HDT1 through HDT60) and ended with a 15-d recovery phase (R+0 through R+14). After the end of each campaign, the participants reported to the laboratory for follow-up examinations. Each campaign included 12 mixed-group participants, however, in the second campaign the number of participants dropped to 11, after one participant dropped out due to medical reasons unrelated to the study. After the BDC phase, participants were randomly divided into two groups: a jump training group (JUMP, n = 12) that performed a specific training protocol (see next section) and a control group (CON, n = 11) that did not perform any exercise during HDT. During the baseline (BDC) and recovery (R) phases, physical activity was limited within the ward in the Envihab. During the HTD phase, all participants maintained a 6° head tilt down position for 24 h/day, while being staff monitored to ensure safety and compliance with the protocol. The permitted range of motion of their heads was up to 30° from horizontal, but static and dynamic muscle contractions were not allowed (except for the planned exercise sessions in the JUMP group). Participants were accommodated in single-person rooms, equipped with television, telephone and laptop with internet access. Room temperature was kept at 20–23 °C, humidity at 30–50%, and the scheduled wake-up was at 6:30 AM, while lights off at 11 PM. All necessary hygiene activities were performed in the HDT position. Redox measurements in plasma and erythrocytes were performed at several time points during all three phases of the study, while urine F2isoprostanes were measured only at baseline (BDC-1) and at the end of the experimental treatment (HDT60) for technical reasons.
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2.3. Familiarization and exercise protocols
approximately 3 pg/mL body fluid.
During the BDC phase, participants were familiarized with the jumping technique in the training device called Sledge Jump System (SJS). The participants completed nine 30-min familiarization sessions with gradually increasing force in the SJS (from 50 to 100% body weight), consisting of a warm-up period, 6 countermovement jumps (CMJ) and 4 series of 10 hops. During the HDT phase, the JUMP group underwent four different training protocols (A, B, C and D in Ref. [38]), completing 48 training sessions in total (6 × type A, 15 × type B, 14 × type C and 13 × type D). Briefly, each session consisted of a warm-up and a varying number of CMJs and repetitive hops of varying intensity (according to the training protocol), as well as three maximal CMJs at 80% body weight. Ground reaction forces as well as sledge position data were recorded for each jump. During the recovery (R) phase, all participants completed six 30-min sessions. Reconditioning consisted of active stretching, fast footwork with an agility ladder, and exercises on a Bosu ball (Bosu, Ashland, Ohio, USA).
2.7. Statistical analysis A two-way repeated measures ANOVA was used to investigate the differences in protein carbonyls, F2-isoprostanes and glutathione content as well as in catalase activity between the two experimental groups (i.e., CON and JUMP) during the study period (BDC, HDT and R phases). When a significant interaction was obtained, pairwise comparisons were performed through the Sidak test. When sphericity was violated, the Greenhouse-Geisser correction was applied. Data are presented as mean ± standard deviation (SD) and the level of significance was set at a = 0.05. 3. Results The repeated measures ANOVA revealed a main effect of time in all dependent variables (P < .05). In particular, plasma protein carbonyls, urine F2-isoprostanes and erythrocyte catalase activity increased, while erythrocyte glutathione concentration decreased over time in both groups. In contrast, neither a main effect of group nor a significant time × group interaction was found in any of the measured variables (P > .05). More specifically and regarding protein carbonyls, a significant main effect of time was found (P = .004). On the contrary, there was no significant main effect of group (P = .492) or time × group interaction (P = .682) (Fig. 2). Regarding GSH, a significant main effect of time (P = .019) was found, but there was no significant main effect of group (P = .629) or time × group interaction (P = .328) (Fig. 3). Likewise, catalase activity showed a significant main effect of time (P = .046) and a non-significant main effect of group (P = .968) or time × group interaction (P = .413) (Fig. 4). Finally, a significant main effect of time was found in F2-isoprostanes (P = .002), while no significant main effect of group (P = .887) or time × group interaction (P = .749) was observed (Fig. 5). All samples from the participants were measured and were equal to n = 11 for the CON group and n = 12 for the JUMP group. The intraand inter-assay coefficient of variations were: 5.3% and 12.1% for protein carbonyls, 5.1% and 13.2% for F2-isoprostanes, 3.3% and 7.9% for GSH and 4.1% and 6.5% for catalase activity (Figs. 2–5).
2.4. Sample collection and medical support Blood and urine sample collection was performed for the assessment of redox biomarkers (i.e., protein carbonyls, F2-isoprostanes, glutathione and catalase) at diverse time points during all phases of the study. Samples were collected in the morning after an overnight fast. Blood samples were obtained from an antecubital vein with a butterfly needle and the Sarstedt blood collection system (Sarstedt AG & Co., Nümbrecht, Germany). To prevent many successive venipunctures, an indwelling venous catheter was used during those project phases that required multiple blood draws. After sample collection, blood was immediately centrifuged at 4 °C and the plasma was separated from packed erythrocytes, which were then lysed with 1:1 (v/v) distilled water. All body fluid samples (blood plasma, erythrocyte lysate and urine) were stored at −80 °C and thawed only once before analysis. A particular manipulation, namely addition of N-ethylmaleimide, was made to the erythrocyte samples that were prepared for the glutathione (GSH) measurement in order to avoid artifactual oxidation to glutathione disulfide (GSSG). Blood pressure, heart rate and body composition were assessed daily, while an independent medical doctor periodically assessed safety parameters. Psychological support talks were conducted weekly by an independent psychologist (for further details on medical support see Ref. [38]).
4. Discussion 2.5. Diet Recently published papers analyzing data from the “Cologne RSL Study” have demonstrated that the eccentric-biased exercise training prevented the 60-d bedrest-induced decrease in maximal knee extension torque, tibial bone mineral content/density and peak oxygen uptake [39], leg power and strength [43], resting muscle tone [44], posture control, gait, and functional mobility [45], muscle oxygen uptake and heart rate kinetics [46] as well as in cardiovascular deconditioning [47]. The aim of the present study was to investigate whether the effects of exercise training, performed using a novel exercise device (i.e., Sledge Jump System) applying eccentric-biased contractions, on head tilt down bed rest-induced muscle atrophy were redox mediated. With respect to muscle atrophy, and according to recently presented data of the Cologne RSL Study, the eccentric-biased exercise training was found to prevent the decrease in lean body mass and lean leg mass elicited during a 60-d bed rest protocol [38,39]. These findings highlighted the importance of muscle loading as a countermeasure against spaceflightrelated declines in muscle mass and function [48]. However, contrary to our hypothesis, this beneficial effect seems to be not mediated by redox processes. In fact, although bed rest disturbed redox homeostasis (as compared to baseline values; BDC), no difference was found between the CON and JUMP groups in two oxidative stress
During the entire study, the subjects’ diet was strictly controlled and individually tailored. The relative intake levels were approximately 14% protein, 35% fat, 49% carbohydrates and 2% fibers. Vitamins and elements were controlled and achieved according to the dietary reference intakes of the National Institutes of Health. The daily water intake was 50 mL/kg body mass. Following demanding physical activity, additional water and diluted apple juice were administered as an offset against sweat and energy loss, whereas, caffeine and alcohol consumption were prohibited. 2.6. Assays Concentration of plasma protein carbonyls and erythrocyte glutathione as well as erythrocyte catalase activity were measured spectrophotometrically as described previously [42]. A competitive immunoassay was used for the quantitation of urine F2-isoprostanes (Cayman Chemical, Charlotte, USA). Urine was purified using the solid phase extraction cartridges. The purification and the subsequent ELISA assay were performed following the manufacturer's recommendations. The sensitivity (80% B/B0) of the immunoassay used is of 118
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Fig. 2. Plasma protein carbonyls in the control (open circles) and exercise (closed circles) groups.
not support these findings, reporting slight or no change in antioxidants (i.e., glutathione and thiol content) during bed rest [51,56] or unaffected DNA oxidation expressed via 8-hydroxy-2′-deoxyguanosine [57]. The authors of the latter study argued that the duration of bed rest protocol (30 days in their work) as a model to simulate spaceflightrelated microgravity might be a critical methodological component to induce relevant redox alterations. Moreover, and in contrast to most studies that evaluated oxidative stress only at two (prior to and after bed rest) or limited time points, we report 9 sampling points, strengthening the observations that bed rest induces oxidative stress. Considering also that the majority of the spaceflight studies and Earthbased analogs demonstrate increased oxidative stress levels during and after prolonged muscle inactivity [58], our oxidative stress findings coincide with the literature. The first report on increased free radical production during exercise goes back to the late 70's [59]. Nowadays, it is well-established that exercise (of any type) represents the most potent physiological redox stimulus lasting from few hours up to 3 days [60]. Interestingly, exercise was for a long time considered exclusively a pro-oxidant stimulus leading transiently to oxidative stress. However, chronic exercise triggers the endogenous antioxidant mechanisms via diverse redox-related
biomarkers (urine F2-isoprostanes and plasma protein carbonyls), in one enzymatic (erythrocyte catalase activity) and one non-enzymatic (glutathione concentration) antioxidant. Our findings also provide some evidence that the concurrent existence of muscle atrophy and oxidative stress during bed rest does not necessarily establish a causeand-effect relationship and/or indicate only a weak connection between them [27]. Another issue worth mentioning is the relatively high variability in redox parameters. This is probably due to the wide redox heterogeneity among individuals, a fact that has been previously highlighted by our group even among individuals of similar age, sex, physical fitness and nutritional habits at rest and in response to an oxidant stimulus [41,49,50]. Of note, this inter-individual variability has been observed both in oxidative stress biomarkers and in enzymatic and low molecular weight antioxidants. Several studies using the bed rest model to simulate space environments have previously measured redox biomarkers. Most of these studies report increased oxidative stress levels and impaired antioxidants concentration in response to bed rest. This is exemplified for instance by increased protein oxidation and lipid peroxidation as well as by decreased low molecular weight antioxidants and altered antioxidant enzyme activity [51–55]. On the other hand, some studies do
Fig. 3. Erythrocyte reduced glutathione in the control (open circles) and exercise (closed circles) groups. 119
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Fig. 4. Erythrocyte catalase activity in the control (open circles) and exercise (closed circles) groups.
Another explanation could be the presence of redox “noise” due to the constantly increased bed rest-induced oxidative stress, which led to a faint exercise-induced redox signal (i.e., masked increases in oxidative stress in response to exercise). We have previously shown that the extent of the acute exercise-induced oxidative stress increases is an important determinant of the redox and physiological adaptations observed after a training protocol [41]. Moreover, we have demonstrated that this extent is largely determined by the baseline levels [67]. Hence, it is reasonable to speculate that our exercise stimulus was not “sensed” by the participants to initiate an adaptive redox response. In this regard, antioxidant treatments to reduce steady state oxidative stress levels in order to augment subsequently the redox signal of exercise could be of value. Nevertheless, this speculative scenario remains elusive and should be experimentally verified. There are some acknowledged limitations in the present study. First,
pathways (e.g., the well-described Keap1-Nrf2-ARE pathway; [61]) driving the expression of target genes related to key antioxidant enzymes, resulting thereby in lower steady state oxidative stress levels [62]. According to this idea, we aimed to use exercise as a redox stimulus to induce repeated transient oxidative stress episodes during the entire bed rest period and we expected to gradually decrease resting levels in the JUMP group in the long term. However, our results did not verify this theoretical framework. In fact, oxidative stress remained increased during the 60 days HTD bed rest in both CON and JUMP groups. A possible explanation for these results is the type of exercise used in this study. In particular, the present protocol is largely eccentric-biased, which has been repeatedly reported to induce potent and long-lasting redox disturbances [49,63–66]. This feature in combination with the persistent redox alterations due to bed rest may have acted synergistically and not antagonistically as hypothesized.
Fig. 5. Urine F2-isoprostanes in the control (open circles) and exercise (closed circles) groups. 120
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we used a battery of redox biomarkers (i.e. a lipid peroxidation and a protein oxidation biomarker, an enzymatic and a non-enzymatic antioxidant). These biomarkers have been criticized as indirect markers of RONS production that do not provide mechanistic insights about the strict and compartmentalized redox signaling pathways that regulate specific biological phenotypes (in our case muscle atrophy). Yet, in the context of the present study, these biomarkers were purely used as “diagnostics” of oxidative stress and to pinpoint a likely redox underlying component [68]. Second, all redox biomarkers were measured in body fluids (i.e., blood and urine) and not in the target tissue (i.e., skeletal muscle). Although it has been demonstrated that blood adequately reflects tissue oxidative stress [69], we cannot safely argue that under extraordinary tissue-specific conditions (e.g., during severe muscle atrophy due to bed rest), a 1:1 link between blood and skeletal muscle exists. Taking also into account the redox “autonomy” of blood, conclusive interpretation from the results presented herein should be drawn with caution. Third, we could not secure samples at all time points for every single biomarker. For instance, F2-isoprostanes were measured prior to and at the end of the HDT period, while protein carbonyls were measured 6 times throughout the same period.
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5. Conclusion Sixty days of 6° head tilt down bed rest (the most frequently used experimental model to simulate microgravity space environment with human subjects) increased systemic oxidative stress and decreased antioxidant levels. These redox alterations were not prevented by exercise despite the fact that lean body mass was preserved. Thus, our findings indicate that the anti-atrophy effects of exercise training during bed rest are not regulated by redox processes, as assessed indirectly via redox biomarkers in blood and urine. Declarations of competing interest None. Acknowledgements The authors would like to thank all of the DLR staff involved in the study and the 23 participants enrolled in the study. The entire Cologne RSL Study was supported by the European Space Agency (400011387115-NL). The construction of the Sledge Jump System was funded by the European Space Agency (ESA Life Sciences Program TEC-MMG/2006/ 82). References [1] T.J. Goodwin, M. Christofidou-Solomidou, Oxidative stress and space biology: an organ-based approach, Int. J. Mol. Sci. 19 (2018) 959, https://doi.org/10.3390/ ijms19040959. [2] J. Guan, X.S. Wan, Z. Zhou, J. Ware, J.J. Donahue, J.E. Biaglow, A.R. Kennedy, Effects of dietary supplements on space radiation-induced oxidative stress in Sprague-Dawley rats, Radiat. Res. 162 (2004) 572–579, https://doi.org/10.1667/ rr3249. [3] A.M. Rizzo, P.A. Corsetto, G. Montorfano, S. Milani, S. Zava, S. Tavella, R. Cancedda, B. Berra, Effects of long-term space flight on erythrocytes and oxidative stress of rodents, PLoS One 7 (2012) e32361, , https://doi.org/10.1371/ journal.pone.0032361. [4] M. Ikemoto, T. Nikawa, M. Kano, K. Hirasaka, T. Kitano, C. Watanabe, R. Tanaka, T. Yamamoto, M. Kamada, K. Kishi, Cysteine supplementation prevents unweighting-induced ubiquitination in association with redox regulation in rat skeletal muscle, Biol. Chem. 383 (2002) 715–721, https://doi.org/10.1515/BC.2002. 074. [5] J.M. Lawler, W. Song, S.R. Demaree, Hindlimb unloading increases oxidative stress and disrupts antioxidant capacity in skeletal muscle, Free Radic. Biol. Med. 35 (2003) 9–16, https://doi.org/10.1016/s0891-5849(03)00186-2. [6] S. Versari, G. Longinotti, L. Barenghi, J.A. Maier, S. Bradamante, The challenging environment on board the International Space Station affects endothelial cell function by triggering oxidative stress through thioredoxin interacting protein overexpression: the ESA-SPHINX experiment, FASEB J. 27 (2013) 4466–4475, https://doi.org/10.1096/fj.13-229195.
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Acta Astronautica journal homepage: www.elsevier.com/locate/actaastro
Research paper
Systemic redox biomarkers suggest non-redox mediated processes in the prevention of bed rest-induced muscle atrophy after exercise training: The Cologne RSL study
T
C.F. Dolopikoua, I.A. Kourtzidisa, A.N. Tsiftsisa,b, N.V. Margaritelisa,b, A.A. Theodorouc, V. Paschalisc,d, C.A. Frantzidise,f, M.G. Nikolaidisa, C. Kourtidou-Papadelif,g, A. Kyparosa,f,∗ a
Department of Physical Education and Sports Science at Serres, Aristotle University of Thessaloniki, 62110, Serres, Greece Intensive Care Unit, 424 General Military Hospital of Thessaloniki, 56429, Thessaloniki, Greece Department of Life Sciences, School of Sciences, European University Cyprus, 1516, Nicosia, Cyprus d School of Physical Education and Sport Science, National and Kapodistrian University of Athens, 17237, Athens, Greece e Laboratory of Medical Physics, Medical School, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece f Greek AeroSpace Medical Association – Space Research (GASMA-SR), 44 Ethnikis Antistasis, 55133, Thessaloniki, Greece g Aeromedical Center of Thessaloniki (AeMC), 44 Ethnikis Antistasis, 55133, Thessaloniki, Greece b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Atrophy Eccentric exercise Muscle Oxidative stress Redox Blood Urine
It has been previously reported that eccentric-biased exercise training prevents the decreases in lean body mass after 60 days of head tilt down bed rest (“Cologne RSL Study”). The aim of the present study, as a part of Cologne RSL Study, was to investigate whether these anti-atrophy effects of exercise training are regulated by redox processes, as assessed indirectly via redox biomarkers in blood and urine. Twenty-four volunteers (N = 24) participated in a randomized controlled study and were randomly divided into two groups: a jump training group (JUMP, n = 12) that performed a specific eccentric-biased training protocol on a Sledge Jump System and a control group (CON, n = 11; one drop-out) that did not perform any exercise. All participants maintained a 6° head tilt down position for 24 h/day for 60 days. Redox measurements in plasma, erythrocytes and urine were performed at several time points throughout the study (i.e., baseline, intervention and recovery phases). A main effect of time was found for all dependent variables (P < .05). In particular, plasma protein carbonyls, erythrocyte catalase activity and urine F2-isoprostanes increased, while erythrocyte glutathione concentration decreased over time in both groups. In contrast, neither a main effect of group nor a significant group × time interaction was found in any of the measured variables (P > .05). In conclusion, our findings in systemic redox biomarkers indicate that the anti-atrophy effects of exercise training during a 60-day bed rest protocol are not regulated by redox processes.
1. Introduction Microgravity is a unique extraterrestrial component of the spaceflight environment, along with cosmic radiation, that severely challenges human health by triggering complex molecular and biochemical redox cascades [1]. Diverse spaceflight and Earth-based analog studies have shown that redox homeostasis is severely disturbed in cells and tissues. In particular, increased oxidative stress levels have been reported during spaceflight as well as in experimental set-ups that aimed to simulated space environments in vitro, ex vivo or in situ, including
various cells and tissues: rat and mouse blood after gamma rays exposure or flight at the International Space Station [2,3], rat skeletal muscle after tail-suspension or hindlimb unloading [4,5], human umbilical vein endothelial cells subjected to flight [6], rat neuronal cells after being seeded onto a high aspect rotating-wall vessel [7], mouse retina [8] and skin [9] after flight at the Space Shuttle Atlantis (STS135), rat liver after flight at STS-63 [10] and mouse brain after hindlimb suspension [11]. Interestingly, it has been demonstrated that the time course of oxidative stress varies during spaceflight, with the return to Earth acting as a late-stage oxidant stimulus [12]. The aberrant redox
∗
Corresponding author. Department of Physical Education and Sports Science at Serres, Aristotle University of Thessaloniki, Agios Ioannis, 62110, Serres, Greece. E-mail addresses: [email protected] (C.F. Dolopikou), [email protected] (I.A. Kourtzidis), [email protected] (A.N. Tsiftsis), [email protected] (N.V. Margaritelis), [email protected] (A.A. Theodorou), [email protected] (V. Paschalis), [email protected] (C.A. Frantzidis), [email protected] (M.G. Nikolaidis), [email protected] (C. Kourtidou-Papadeli), [email protected] (A. Kyparos). https://doi.org/10.1016/j.actaastro.2019.12.002 Received 6 October 2019; Received in revised form 29 November 2019; Accepted 2 December 2019 Available online 05 December 2019 0094-5765/ © 2019 IAA. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. The study design of the Cologne RSL Study. BDC: baseline data collection; HDT: head-down tilt position; R: recovery phase.
2. Materials and methods
homeostasis in response to microgravity is also exemplified by the decreased expression and/or activity of major antioxidant enzymes [13–15], the increased ROS production in cell lines [7,16,17], the impaired cellular response to a redox stimulus [18] and the decreased levels of non-enzymatic antioxidants in blood and tissues [11,19–21]. Microgravity also conveys many challenges to the human physiology affecting almost all organs and systems. Taking into account that spaceflights frequently last up to few months, these challenges have to be addressed. Muscle atrophy and the associated loss of muscle force and function have been recognized as a major threat during spaceflight [22,23]. Despite the incredible physical fitness of astronauts, muscle atrophy is one of the earliest complications observed under microgravity. From a redox perspective, it has been hypothesized that the pro-oxidative environment of skeletal muscle exacerbates atrophy via massive oxidation of contractile proteins leading to the activation of the proteolytic mechanisms [24], while nitrosative stress has also been reported to contribute via S-nitrosylation of contractile and other functional proteins in atrophic muscles [25]. Nevertheless, whether oxidative stress is the cause, the consequence or just an epiphenomenon of skeletal muscle atrophy, especially during spaceflight, remains still elusive [26,27]. Bed rest in a head-down tilt position and the hind-limb unloading method represent the two most frequently used models to simulate microgravity conditions. Both conditions result to a cephalad fluid shift and a severe decline in mechanical loading of skeletal muscles, two fundamental characteristics of microgravity. However, the bed rest model is most frequently applied in studies with human subjects compared to the hindlimb unloading model which is typically used in studies with rodents [28,29]. There are several studies that used the bed rest model for prolonged periods equal to 90 [30], 60 [31,32], 55 [33], 35 [34] and 14 days [35]. There are also some papers indicating that only a week [36] or even 5 days [37] of bed rest are enough to facilitate protein degradation and hence lead to muscle atrophy. Recently published data of the “Cologne RSL Study” demonstrated that the eccentric-biased exercise training prevented the decrease in lean body mass and lean leg mass elicited during a 60-d bed rest protocol [38,39]. Exercise as a redox stimulus transiently increases free radical production, yet in the long term it upregulates antioxidant mechanisms and decreases resting oxidative stress levels [40]. Taking also into account that exercise-induced oxidative stress is an essential signal for adaptations [41], it would be interesting to know if the effect of exercise as a countermeasure against the bed rest-induced muscle atrophy is redox-mediated. Thus, the aim of the present study, as part of the Cologne RSL Study, was to investigate whether the anti-atrophy effects (reported in Refs. [38,39]) of eccentric-biased exercise training during 60 days of 6° head tilt down bed rest are mediated by redox processes, as assessed indirectly via redox biomarkers in two different body fluids (i.e., blood and urine).
2.1. Participants Twenty-four male non-smoker adults aged 20–45 years old, with a body mass index between 20 and 26 kg/m2 were selected among 494 original volunteers based on specific physiological, psychological and medical inclusion criteria, described in detail by Kramer et al. [38]. All participants gave written informed consent to participate in the experimental procedures, which were approved by the Ethics Committee of the Northern Rhine Medical Association (Ärztekammer Nordrhein) in Düsseldorf, Germany, and the Federal Office for Radiation Protection (Bundesamt für Strahlenschutz). Participants received a financial reward for participating in the study.
2.2. Study design The volunteers (N = 24) participated in a randomized controlled training study, conducted at the Envihab, a medical research facility of the Institute of Aerospace Medicine at the German Aerospace Center (DLR) in Cologne, Germany. The study was accomplished in 2 campaigns, the first one taking place in August 2015 and the second one in January 2016. The entire study design is illustrated in Fig. 1. Each campaign lasted 90 days; 15 days of baseline data collection (BDC-15 through BDC-1), followed by a 60-d intervention phase (HDT1 through HDT60) and ended with a 15-d recovery phase (R+0 through R+14). After the end of each campaign, the participants reported to the laboratory for follow-up examinations. Each campaign included 12 mixed-group participants, however, in the second campaign the number of participants dropped to 11, after one participant dropped out due to medical reasons unrelated to the study. After the BDC phase, participants were randomly divided into two groups: a jump training group (JUMP, n = 12) that performed a specific training protocol (see next section) and a control group (CON, n = 11) that did not perform any exercise during HDT. During the baseline (BDC) and recovery (R) phases, physical activity was limited within the ward in the Envihab. During the HTD phase, all participants maintained a 6° head tilt down position for 24 h/day, while being staff monitored to ensure safety and compliance with the protocol. The permitted range of motion of their heads was up to 30° from horizontal, but static and dynamic muscle contractions were not allowed (except for the planned exercise sessions in the JUMP group). Participants were accommodated in single-person rooms, equipped with television, telephone and laptop with internet access. Room temperature was kept at 20–23 °C, humidity at 30–50%, and the scheduled wake-up was at 6:30 AM, while lights off at 11 PM. All necessary hygiene activities were performed in the HDT position. Redox measurements in plasma and erythrocytes were performed at several time points during all three phases of the study, while urine F2isoprostanes were measured only at baseline (BDC-1) and at the end of the experimental treatment (HDT60) for technical reasons.
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2.3. Familiarization and exercise protocols
approximately 3 pg/mL body fluid.
During the BDC phase, participants were familiarized with the jumping technique in the training device called Sledge Jump System (SJS). The participants completed nine 30-min familiarization sessions with gradually increasing force in the SJS (from 50 to 100% body weight), consisting of a warm-up period, 6 countermovement jumps (CMJ) and 4 series of 10 hops. During the HDT phase, the JUMP group underwent four different training protocols (A, B, C and D in Ref. [38]), completing 48 training sessions in total (6 × type A, 15 × type B, 14 × type C and 13 × type D). Briefly, each session consisted of a warm-up and a varying number of CMJs and repetitive hops of varying intensity (according to the training protocol), as well as three maximal CMJs at 80% body weight. Ground reaction forces as well as sledge position data were recorded for each jump. During the recovery (R) phase, all participants completed six 30-min sessions. Reconditioning consisted of active stretching, fast footwork with an agility ladder, and exercises on a Bosu ball (Bosu, Ashland, Ohio, USA).
2.7. Statistical analysis A two-way repeated measures ANOVA was used to investigate the differences in protein carbonyls, F2-isoprostanes and glutathione content as well as in catalase activity between the two experimental groups (i.e., CON and JUMP) during the study period (BDC, HDT and R phases). When a significant interaction was obtained, pairwise comparisons were performed through the Sidak test. When sphericity was violated, the Greenhouse-Geisser correction was applied. Data are presented as mean ± standard deviation (SD) and the level of significance was set at a = 0.05. 3. Results The repeated measures ANOVA revealed a main effect of time in all dependent variables (P < .05). In particular, plasma protein carbonyls, urine F2-isoprostanes and erythrocyte catalase activity increased, while erythrocyte glutathione concentration decreased over time in both groups. In contrast, neither a main effect of group nor a significant time × group interaction was found in any of the measured variables (P > .05). More specifically and regarding protein carbonyls, a significant main effect of time was found (P = .004). On the contrary, there was no significant main effect of group (P = .492) or time × group interaction (P = .682) (Fig. 2). Regarding GSH, a significant main effect of time (P = .019) was found, but there was no significant main effect of group (P = .629) or time × group interaction (P = .328) (Fig. 3). Likewise, catalase activity showed a significant main effect of time (P = .046) and a non-significant main effect of group (P = .968) or time × group interaction (P = .413) (Fig. 4). Finally, a significant main effect of time was found in F2-isoprostanes (P = .002), while no significant main effect of group (P = .887) or time × group interaction (P = .749) was observed (Fig. 5). All samples from the participants were measured and were equal to n = 11 for the CON group and n = 12 for the JUMP group. The intraand inter-assay coefficient of variations were: 5.3% and 12.1% for protein carbonyls, 5.1% and 13.2% for F2-isoprostanes, 3.3% and 7.9% for GSH and 4.1% and 6.5% for catalase activity (Figs. 2–5).
2.4. Sample collection and medical support Blood and urine sample collection was performed for the assessment of redox biomarkers (i.e., protein carbonyls, F2-isoprostanes, glutathione and catalase) at diverse time points during all phases of the study. Samples were collected in the morning after an overnight fast. Blood samples were obtained from an antecubital vein with a butterfly needle and the Sarstedt blood collection system (Sarstedt AG & Co., Nümbrecht, Germany). To prevent many successive venipunctures, an indwelling venous catheter was used during those project phases that required multiple blood draws. After sample collection, blood was immediately centrifuged at 4 °C and the plasma was separated from packed erythrocytes, which were then lysed with 1:1 (v/v) distilled water. All body fluid samples (blood plasma, erythrocyte lysate and urine) were stored at −80 °C and thawed only once before analysis. A particular manipulation, namely addition of N-ethylmaleimide, was made to the erythrocyte samples that were prepared for the glutathione (GSH) measurement in order to avoid artifactual oxidation to glutathione disulfide (GSSG). Blood pressure, heart rate and body composition were assessed daily, while an independent medical doctor periodically assessed safety parameters. Psychological support talks were conducted weekly by an independent psychologist (for further details on medical support see Ref. [38]).
4. Discussion 2.5. Diet Recently published papers analyzing data from the “Cologne RSL Study” have demonstrated that the eccentric-biased exercise training prevented the 60-d bedrest-induced decrease in maximal knee extension torque, tibial bone mineral content/density and peak oxygen uptake [39], leg power and strength [43], resting muscle tone [44], posture control, gait, and functional mobility [45], muscle oxygen uptake and heart rate kinetics [46] as well as in cardiovascular deconditioning [47]. The aim of the present study was to investigate whether the effects of exercise training, performed using a novel exercise device (i.e., Sledge Jump System) applying eccentric-biased contractions, on head tilt down bed rest-induced muscle atrophy were redox mediated. With respect to muscle atrophy, and according to recently presented data of the Cologne RSL Study, the eccentric-biased exercise training was found to prevent the decrease in lean body mass and lean leg mass elicited during a 60-d bed rest protocol [38,39]. These findings highlighted the importance of muscle loading as a countermeasure against spaceflightrelated declines in muscle mass and function [48]. However, contrary to our hypothesis, this beneficial effect seems to be not mediated by redox processes. In fact, although bed rest disturbed redox homeostasis (as compared to baseline values; BDC), no difference was found between the CON and JUMP groups in two oxidative stress
During the entire study, the subjects’ diet was strictly controlled and individually tailored. The relative intake levels were approximately 14% protein, 35% fat, 49% carbohydrates and 2% fibers. Vitamins and elements were controlled and achieved according to the dietary reference intakes of the National Institutes of Health. The daily water intake was 50 mL/kg body mass. Following demanding physical activity, additional water and diluted apple juice were administered as an offset against sweat and energy loss, whereas, caffeine and alcohol consumption were prohibited. 2.6. Assays Concentration of plasma protein carbonyls and erythrocyte glutathione as well as erythrocyte catalase activity were measured spectrophotometrically as described previously [42]. A competitive immunoassay was used for the quantitation of urine F2-isoprostanes (Cayman Chemical, Charlotte, USA). Urine was purified using the solid phase extraction cartridges. The purification and the subsequent ELISA assay were performed following the manufacturer's recommendations. The sensitivity (80% B/B0) of the immunoassay used is of 118
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Fig. 2. Plasma protein carbonyls in the control (open circles) and exercise (closed circles) groups.
not support these findings, reporting slight or no change in antioxidants (i.e., glutathione and thiol content) during bed rest [51,56] or unaffected DNA oxidation expressed via 8-hydroxy-2′-deoxyguanosine [57]. The authors of the latter study argued that the duration of bed rest protocol (30 days in their work) as a model to simulate spaceflightrelated microgravity might be a critical methodological component to induce relevant redox alterations. Moreover, and in contrast to most studies that evaluated oxidative stress only at two (prior to and after bed rest) or limited time points, we report 9 sampling points, strengthening the observations that bed rest induces oxidative stress. Considering also that the majority of the spaceflight studies and Earthbased analogs demonstrate increased oxidative stress levels during and after prolonged muscle inactivity [58], our oxidative stress findings coincide with the literature. The first report on increased free radical production during exercise goes back to the late 70's [59]. Nowadays, it is well-established that exercise (of any type) represents the most potent physiological redox stimulus lasting from few hours up to 3 days [60]. Interestingly, exercise was for a long time considered exclusively a pro-oxidant stimulus leading transiently to oxidative stress. However, chronic exercise triggers the endogenous antioxidant mechanisms via diverse redox-related
biomarkers (urine F2-isoprostanes and plasma protein carbonyls), in one enzymatic (erythrocyte catalase activity) and one non-enzymatic (glutathione concentration) antioxidant. Our findings also provide some evidence that the concurrent existence of muscle atrophy and oxidative stress during bed rest does not necessarily establish a causeand-effect relationship and/or indicate only a weak connection between them [27]. Another issue worth mentioning is the relatively high variability in redox parameters. This is probably due to the wide redox heterogeneity among individuals, a fact that has been previously highlighted by our group even among individuals of similar age, sex, physical fitness and nutritional habits at rest and in response to an oxidant stimulus [41,49,50]. Of note, this inter-individual variability has been observed both in oxidative stress biomarkers and in enzymatic and low molecular weight antioxidants. Several studies using the bed rest model to simulate space environments have previously measured redox biomarkers. Most of these studies report increased oxidative stress levels and impaired antioxidants concentration in response to bed rest. This is exemplified for instance by increased protein oxidation and lipid peroxidation as well as by decreased low molecular weight antioxidants and altered antioxidant enzyme activity [51–55]. On the other hand, some studies do
Fig. 3. Erythrocyte reduced glutathione in the control (open circles) and exercise (closed circles) groups. 119
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Fig. 4. Erythrocyte catalase activity in the control (open circles) and exercise (closed circles) groups.
Another explanation could be the presence of redox “noise” due to the constantly increased bed rest-induced oxidative stress, which led to a faint exercise-induced redox signal (i.e., masked increases in oxidative stress in response to exercise). We have previously shown that the extent of the acute exercise-induced oxidative stress increases is an important determinant of the redox and physiological adaptations observed after a training protocol [41]. Moreover, we have demonstrated that this extent is largely determined by the baseline levels [67]. Hence, it is reasonable to speculate that our exercise stimulus was not “sensed” by the participants to initiate an adaptive redox response. In this regard, antioxidant treatments to reduce steady state oxidative stress levels in order to augment subsequently the redox signal of exercise could be of value. Nevertheless, this speculative scenario remains elusive and should be experimentally verified. There are some acknowledged limitations in the present study. First,
pathways (e.g., the well-described Keap1-Nrf2-ARE pathway; [61]) driving the expression of target genes related to key antioxidant enzymes, resulting thereby in lower steady state oxidative stress levels [62]. According to this idea, we aimed to use exercise as a redox stimulus to induce repeated transient oxidative stress episodes during the entire bed rest period and we expected to gradually decrease resting levels in the JUMP group in the long term. However, our results did not verify this theoretical framework. In fact, oxidative stress remained increased during the 60 days HTD bed rest in both CON and JUMP groups. A possible explanation for these results is the type of exercise used in this study. In particular, the present protocol is largely eccentric-biased, which has been repeatedly reported to induce potent and long-lasting redox disturbances [49,63–66]. This feature in combination with the persistent redox alterations due to bed rest may have acted synergistically and not antagonistically as hypothesized.
Fig. 5. Urine F2-isoprostanes in the control (open circles) and exercise (closed circles) groups. 120
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we used a battery of redox biomarkers (i.e. a lipid peroxidation and a protein oxidation biomarker, an enzymatic and a non-enzymatic antioxidant). These biomarkers have been criticized as indirect markers of RONS production that do not provide mechanistic insights about the strict and compartmentalized redox signaling pathways that regulate specific biological phenotypes (in our case muscle atrophy). Yet, in the context of the present study, these biomarkers were purely used as “diagnostics” of oxidative stress and to pinpoint a likely redox underlying component [68]. Second, all redox biomarkers were measured in body fluids (i.e., blood and urine) and not in the target tissue (i.e., skeletal muscle). Although it has been demonstrated that blood adequately reflects tissue oxidative stress [69], we cannot safely argue that under extraordinary tissue-specific conditions (e.g., during severe muscle atrophy due to bed rest), a 1:1 link between blood and skeletal muscle exists. Taking also into account the redox “autonomy” of blood, conclusive interpretation from the results presented herein should be drawn with caution. Third, we could not secure samples at all time points for every single biomarker. For instance, F2-isoprostanes were measured prior to and at the end of the HDT period, while protein carbonyls were measured 6 times throughout the same period.
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5. Conclusion Sixty days of 6° head tilt down bed rest (the most frequently used experimental model to simulate microgravity space environment with human subjects) increased systemic oxidative stress and decreased antioxidant levels. These redox alterations were not prevented by exercise despite the fact that lean body mass was preserved. Thus, our findings indicate that the anti-atrophy effects of exercise training during bed rest are not regulated by redox processes, as assessed indirectly via redox biomarkers in blood and urine. Declarations of competing interest None. Acknowledgements The authors would like to thank all of the DLR staff involved in the study and the 23 participants enrolled in the study. The entire Cologne RSL Study was supported by the European Space Agency (400011387115-NL). The construction of the Sledge Jump System was funded by the European Space Agency (ESA Life Sciences Program TEC-MMG/2006/ 82). References [1] T.J. Goodwin, M. Christofidou-Solomidou, Oxidative stress and space biology: an organ-based approach, Int. J. Mol. Sci. 19 (2018) 959, https://doi.org/10.3390/ ijms19040959. [2] J. Guan, X.S. Wan, Z. Zhou, J. Ware, J.J. Donahue, J.E. Biaglow, A.R. Kennedy, Effects of dietary supplements on space radiation-induced oxidative stress in Sprague-Dawley rats, Radiat. Res. 162 (2004) 572–579, https://doi.org/10.1667/ rr3249. [3] A.M. Rizzo, P.A. Corsetto, G. Montorfano, S. Milani, S. Zava, S. Tavella, R. Cancedda, B. Berra, Effects of long-term space flight on erythrocytes and oxidative stress of rodents, PLoS One 7 (2012) e32361, , https://doi.org/10.1371/ journal.pone.0032361. [4] M. Ikemoto, T. Nikawa, M. Kano, K. Hirasaka, T. Kitano, C. Watanabe, R. Tanaka, T. Yamamoto, M. Kamada, K. Kishi, Cysteine supplementation prevents unweighting-induced ubiquitination in association with redox regulation in rat skeletal muscle, Biol. Chem. 383 (2002) 715–721, https://doi.org/10.1515/BC.2002. 074. [5] J.M. Lawler, W. Song, S.R. Demaree, Hindlimb unloading increases oxidative stress and disrupts antioxidant capacity in skeletal muscle, Free Radic. Biol. Med. 35 (2003) 9–16, https://doi.org/10.1016/s0891-5849(03)00186-2. [6] S. Versari, G. Longinotti, L. Barenghi, J.A. Maier, S. Bradamante, The challenging environment on board the International Space Station affects endothelial cell function by triggering oxidative stress through thioredoxin interacting protein overexpression: the ESA-SPHINX experiment, FASEB J. 27 (2013) 4466–4475, https://doi.org/10.1096/fj.13-229195.
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