Muscle Biopsy - MBP - Cell signalling molecules of human skeletal muscle maintained by exercise countermeasure in spaceflight
  1. 2015 • ISS 44S (Soyuz TMA-18M) "IrISS" - short-duration mission
  2. 2015 • ISS Increments 45-46
  3. 2016 • ISS Increments 47-48
  4. 2016 • ISS Increments 49-50
  5. 2017 • ISS Increments 51-52
  6. 2017 • ISS Increments 53-54
  7. 2018 • ISS "Horizons" - long-duration mission
  8. 2018 • ISS Increments 57-58
Life Sciences:
  • Human Physiology
  • Muscle/skeletal system
D. Blottner (1), B.G.H. Schoser (2), M. Salanova (3)
Charité Universitätsmedizin Berlin
Department of Vegetative Anatomy
Center of Space Medicine Berlin (ZWMB)
Neuromuscular System
Philippsstrasse 12
10115 Berlin
Friedrich-Baur Institute
Ziemssenstrasse 1A
80336 München
Charité Universitätsmedizin Berlin
Center of Space Medicine Berlin
Arnimallee 22
14195 Berlin
NOTE: Andreas Mogensen became Denmark’s first astronaut when he launched on board Soyuz TMA-18M (ISS 44S) on 2 September 2015 for his 10-day iriss mission. He landed with Soyuz TMA-16M on 12 September 2015.


Skeletal muscle functions are mediated by cellular signalling mechanisms that may undergo considerable changes due to muscle activity or inactivity on Earth. Nitric oxide (NO) generated by NO-synthase (NOS) is a unique signalling molecule mediating basic physiological muscle functions including microperfusion capacity, soreness and muscle force generation capacity. As demonstrated now in long-term (90d and 60d) bed rest studies in men and in women (LTBR, 2001; BBR, 2003; WISE 2005, BBR-2 2008), resistive training using the flywheel device (FWED), FWED in combination with low-body negative pressure (LBNP), and resistive muscle vibration (Galileo Space device) maintained muscle force and power output as well as cellular and biochemical NOS levels.

Only recently it was showed that compared to resistive-only (RE) exercise countermeasure, vibration exercise (RVE) superimposed to RE training during long-term bed rest (BBR2) is actually based on neurophysiological stimulation via nerve-muscle interaction and that Homer proteins are important molecular players in the neuromuscular adaptation mechanisms to disuse or exercise (Salanova et al., 2012). More importantly, confirmation of severe atrophy marker alterations including NOS changes in skeletal muscle were confirmed in ground-based studies (tissue-sharing) as well as in animal experimentation in real microgravity by the NASA/ASI Mouse Drawer System (MDS) mission (91 days on ISS, NASA STS-129) for example confirming the relocalization of membrane/cytosolic NOS in mouse postural muscles (Sandona D, Desaphy JF et al., PLoS ONE 7:e33232). More recently, the role of Homer proteins in skeletal muscle and neuromuscular adaptation (Figure 3) has been reviewed (Salanova et al., 2013a) and the changes in the NO-linked nitrosative stress management in human skeletal muscle has been analysed for the first time in chronically disused muscle that can be affected by exercise as countermeasure (Salanova et al., 2013b). NOS/NO signalling and nitrosative stress management (Figure 4) are therefore linked to muscle activity in both humans and rodent animals , it can be restored together with other important signalling molecules (Homer) by resistive exercise / vibration in muscle and at the nerve-muscle interface (neuromuscular junction), and thus may serve as molecular biomarker for testing efficacy of countermeasure protocols to support human skeletal muscle performance capacity under real microgravity conditions.

A. Immunolocalization of NOS1-3 isoforms at functional compartments and biochemical analysis of NOS 1-3 and related pathway molecules (Homer, mTor, others) analyzed before and after spaceflight, i.e. pre- and post-flight biopsies, of mixed/fast and slow-type human skeletal muscle.

B. To study altered expression patterns of the muscular NOS/NO system and related molecules (Capon, PIN, immediate early genes, Homer) following countermeasure after short-term exposure to microgravity.

C. To study altered expression patterns of the muscular NOS/NO system and related molecules (Capon, PIN, IEGs, Homer) following countermeasure after long-term exposure to microgravity.
Previous Flight Experiments
MDS mission (STS-129) with mice on ISS (PI R. Cancedda, ASI)

Justification for Need of Space Experiment
A number of ground-based studies including several long-term bed rest studies (and one MTBR presently ongoing at DLR/Cologne) and animal experimentation (MIS project) have been performed or are almost completed. More importantly, microgravity-related data sets from animal experimentation on the ISS (MDS mission STS-128 and STS-129, Sandona et al., 2012) largely confirmed most of our ground-based data sets, and it appears now also obvious that cage area restrictions (e.g. 50% reduced cage area during MDS housing in Space or MIS housing on ground) may not have, on its own, affected the outcome of musculoskeletal and behavioural biological parameters (Blottner et al., 2009) suggesting no major impact of e.g. restricted area animal housing in Space on the scientific outcome of Life Sciences Experimentation. In humans, further planned investigations will show if muscular NOS expression and related molecules will be affected by exposure to actual microgravity in spaceflight and if signalling molecules like NOS and associated pathway molecules can be used in muscle as molecular cell marker to evaluate inflight countermeasure protocols of astronauts to prevent muscle atrophy and thus impaired performance control during extended space missions. The project will be able to collect fundamental data sets of microgravity-induced changes of muscle specific molecules from humans in spaceflight thus complementing the already existing human and animal groundbased data sets from previous work over the last 10 years.
The experiment is a ground-based experiment.


Biopsies (min. 30-50 mg) from fast (m. vastus lateralis) and/or slow-type (m. soleus) human skeletal muscle will be taken from astronauts from one leg only (right leg preferred) before and after spaceflight.
The pre-flight biopsy needs to be taken between L-120 and launch.
It is desired to have the post-flight biopsies before 28 hours after landing, and absolutely not later than 36 hours after.

Parameters Measured

NOS1,-2,-3 gene analysis
NOS1,-2,-3 immuno-histochemistry
NOS associated molecules (CAPON and PIN)
Fiber size
Fiber type

Test Subjects

Number of subjects desired: 8
Number of subjects required: 4
Subject exclusions: n/a

Mission Duration
Minimum Mission Duration: Short flight duration of approx. 10 days is acceptable
Maximum Mission Duration: n/a

Ground Reference Experiments
Ground-based bed rest experiments (in particular short- and medium term studies of e.g. 7-21 days duration, but also long-term 60-90 days) using identical countermeasure protocols and biopsies will run prior to, simultaneously to, and/or post-flight as ground reference for scientific reasons.

Baseline Data Collection
Temperature requirements: all biopsy samples shall be stored frozen in fl. N2 and/or minus -80°C freezer (prior to transport to lab)

Pre-flight: Pre-flight muscle biopsy shall be taken between L-120 and launch.

Post-flight: Post-flight muscle biopsy as soon as possible after landing i.e., 28 hour post landing should be the latest time point, on which muscle biopsies must be sampled from the subjects. Immediate deep freezing of muscle biopsies (within 1-3 min.) in fl. N2 after sampling is strongly required

Early Post-flight Requirements (R+0d to R+4d) justification: All post-flight biopsies have to be taken shortly after g-transition and landing (R+0-1d) as 1g-reloading of muscle is known to seriously affect the muscle tissue and in particular the structure and molecular composition of muscle cells that are reloaded by 1G levels.

Scheduling and session constrains
For pre-flight muscle biopsy:
- No strenuous exercises (above 75% VO2 max) during the 24-hours prior to the biopsy
- No exercises on the day of the biopsy, 4 hours prior to the biopsy
- Caffeine consumption is allowed to crews usual amount (if crew member drinks a cup of coffee every morning, that is okay, but no “extra” caffeine)
- No alcohol 12 hours prior to BDC
For post-flight muscle biopsy:
- No exercises on the day of the biopsy, 4 hours prior to the biopsy
- Caffeine consumption is allowed to crews usual amount (if crew member drinks a cup of coffee every morning, that is okay, but no “extra” caffeine)
- No alcohol 12 hours prior to BDC
- It is desired to have the postflight biopsies before 28 hours after landing, and absolutely not later than 36 hours after.

In addition to NOS gene analysis, NOS1-3 immunohistochemistry (including NOS associated molecules like CAPON and PIN), and all quantitative biochemical analysis will be performed at the laboratories of the Center of Space Medicine Berlin, Germany according to well-established procedures tested for human skeletal muscle. Fixation conditions, dilutions of primary and secondary mono- and polyclonal antibodies, immunostaining procedures and fiber typing are well-established for normal human skeletal muscle tissue and adaptation. All immunofluorescence analysis will be made by light- and epifluorescence microscopy, and by high-resolution confocal laser scanning microscopy using state-of-the art digitized imaging. Analysis also include morphometric measurements of myofiber size, determination of myofiber types (I or II) and morphometric fiber-to-capillary ratio.
Expected Results and Hypothesis:
The first and major goal will be to collect, analyse and interpret data of NOS expression in fast and slow-type human skeletal muscle before spaceflight (ground reference control) and following exposition to actual microgravity in space (microgravity exposure). Results will show if, and to what extent, NOS expression or related molecules will be altered in human skeletal muscle exposed to real microgravity conditions.
Aims B and C are expected to be achieved by comparison of pre vs. post-flight data on NOS/NO system alterations after short- and long-term exposure to microgravity.
It is also expected to document significant effects of exercise countermeasure protocols on NOS expression during prolonged microgravity conditions, that will be used for evaluation of the effectiveness of protocols to support muscle atrophy and to develop reliable biomarkers monitoring molecular changes of the NOS/NO signalling pathway at the cellular level, thus reflecting skeletal muscle function in spaceflight conditions.
M. Salanova, G. Schiffl, B. Püttmann, B.G. Schoser, D. Blottner, (2008), "Molecular biomarkers monitoring human skeletal muscle fibres and micro-vasculature following long-term bed rest with and without countermeasure", Journal of Anatomy, 212, pp. 306-318.
D. Blottner, B. Püttmann, M. Salanova, G. Schiffl, J. Rittweger, H.C. Gunga, D. Felsenberg, B.G. Schoser, (2006), "Skeletal muscle deconditioning, nitric oxide (NO) biomarker, and exercise countermeasure - Five years of bed rest studies", Journal of Gravitational Physiology, 13, 2, pp. 49-58.
D. Blottner, M. Salanova, B. Püttmann, G. Schiffl, D. Felsenberg, J. Rittweger, (2006), "Human skeletal muscle structure and function preserved by vibration muscle exercise following 55-days of bed rest", European Journal of Applied Physiology, 97, pp. 261-271.
A. Moukhina, B. Shenkman, D. Blottner, T. Nemirovskaya, Y. Lemesheva, B. Püttmann, I. Kozlovskaya, (2004), "Effects of support stimulation on human soleus fiber characteristics during exposure to "dry" immersion", Journal of Gravitational Physiology, 11, pp. P137-8.
J. Rudnick, B. Puttmann, P.A. Tesch, B. Alkner, B.G. Schoser, M. Salanova, K. Kirsch, H.C. Gunga, G. Schiffl, G. Lück, D. Blottner, (2004), "Differential expression of nitric oxide synthases (NOS 1-3) in human skeletal muscle following exercise countermeasure during 12 weeks of bed rest", FASEB Journal, 18, pp. 1228-1230.
M. Salanova, G. Schiffl, J. Rittweger, D. Felsenberg, D. Blottner, (2008), "Ryanodine receptor type-1 (RyR1) expression and protein S-nitrosylation pattern in human soleus myofibres following bed rest and exercise countermeasure", Histochemistry and cell biology, 130, 1, pp. 105-118.
M. Salanova, G. Schiffl, D. Blottner, (2009), "Atypical fast SERCA1a protein expression in slow myofibers and differential S-nitrosylation prevented by exercise during long term bed rest", Histochemistry and Cell Biology, 132, 4, pp. 383-394.
D. Blottner, N. Serradj, M. Salanova, C. Touma, R. Palme, M. Silva, J.M. Aerts, D. Berckmans, L. Vico, Y. Liu, A. Giuliani, F. Rustichelli, R. Cancedda, M. Jamon, (2009), "Morphological, physiological and behavioural evaluation of a Mice in Space housing system", Journal of Comparative Physiology B, 179, 4, doi: 10.1007/s00360-008-0330-4, pp. 519-533.
D.L. Belavý, O. Bock, H. Börst, G. Armbrecht, U. Gast, C. Degner, G. Beller, H. Soll, M. Salanova, H. Habazettl, M. Heer, A. de Haan, D.F. Stegeman, P. Cerretelli, D. Blottner, J. Rittweger, C. Gelfi, U. Kornak, D. Felsenberg, (2010), "The 2nd Berlin BedRest Study: protocol and implementation", Journal of Musculoskeletal and Neuronal Interactions - JMNI, 10, 3, pp. 207-219.
M. Moriggi, M. Vasso, C. Fania, D. Capitanio, G. Bonifacio, M. Salanova, D. Blottner, J. Rittweger, D. Felsenberg, P. Cerretelli, C. Gelfi, (2010), "Long-term bed rest with and without vibration exercise countermeasure: effects on human muscle protein dysregulation", Proteomics, 10, 21, doi: 10.1002/pmic.200900817, pp. 3756-3774.
M. Salanova, E. Bortoloso, G. Schiffl, M. Gutsmann, D.L. Belavy, D. Felsenberg, S. Furlan, P. Volpe, D. Blottner, (2011), "Expression and regulation of Homer in human skeletal muscle during neuromuscular junction adaptation to disuse and exercise", FASEB Journal, 25, 12, doi: 10.1096/fj.11-186049, pp. 4312-4325.
D. Sandonà, J.F. Desaphy, G.M. Camerino, E. Bianchini, S. Ciciliot, D. Danieli-Betto, G. Dobrowolny, S. Furlan, E. Germinario, K. Goto, M. Gutsmann, F. Kawano, N. Nakai, T. Ohira, Y. Ohno, A. Picard, M. Salanova, G. Schiffl, D. Blottner, A. Musarò, Y. Ohira, R. Betto, D. Conte, S. Schiaffino, (2012), "Adaptation of mouse skeletal muscle to long-term microgravity in the MDS mission", PLoS ONE, 7, 3, doi: 10.1371/journal.pone.0033232, pp. e33232.
M. Salanova, P. Volpe, D. Blottner, (2013), "Homer family regulation in skeletal muscle and neuromuscular adaptation", International Union of Biochemistry and Molecular Biology Life - IUBMB Life, 65, 9, doi: 10.1002/iub.1198, pp. 769-776.
M. Salanova, G. Schiffl, M. Gutsmann, D. Felsenberg, S. Furlan, P. Volpe, A. Clarke, D. Blottner, (2013), "Nitrosative stress in human skeletal muscle attenuated by exercise countermeasure after chronic disuse", Redox Biology, 1, doi: 10.1016/j.redox.2013.10.006, pp. 514-526.
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Figure 1: Confocal images showing activity-induced plasma membrane expression of nitric oxide synthase type-1 (NOS1, green immunofluorescence) at plasma membranes of crosssectioned biopsy samples of (A) inactively vs. (B) trained human m. soleus myofibers following extended (60 d) bed rest. (C) Without exercise (Control), NOS membrane expression is significantly decreased after bed rest. After bed rest and exercise countermeasure, NOS1 is highly preserved in both slow and fast myofibers (1 and 2). (D) Western immunoblot analysis showing altered NOS1 protein content in human skeletal muscle before (Pre) and after (Post) bed rest (exercise group). compare reference documents 3 and 5

Figure 2: Confocal microscopy showing similar distribution patterns of slow type-1 (dark) and fast type-2 (red) myofibers in (A) normal and (B) exercised human calf m. soleus following long-term (60d) bed rest. (C) Quantitative bar graph showing that the typical slow-to-fast fiber-shift (slow < fast type) of unloaded human m.soleus (Control) is prevented by exercise countermeasure (Exercise) during bed rest suggesting phenotype preservation of the soleus calf muscle. compare reference documents 3 and 5

Figure 3: Expression of Homer (green label) in the subsynaptic area of a human neuromuscular junction following exercise countermeasure. Homer proteins are regulated by motor nerve activity and help to control intramyocellular calcium balance. compare reference documents 11 and 13

Figure 4: Sketch showing nitrosative stress in human skeletal muscle via s-nitrosylated functional protein (SNO-proteins) analysis attenuated by exercise countermeasure following chronic disuse. Chronic disuse (60 days bed rest) results in excess levels of calcium-signaling SNO-proteins (RyR1, SERCA, PMCA) in leg skeletal muscle that can be normalized to baseline by exercise as countermeasure. compare reference document 14

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