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Inhibition of myostatin prevents microgravity-induced loss of skeletal muscle mass and strength

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The microgravity conditions of prolonged spaceflight are known to result in skeletal muscle atrophy that leads to diminished functional performance. To assess if inhibition of the growth factor myostatin has potential to reverse these effects, mice were treated with a myostatin antibody while housed on the International Space Station. Grip strength of ground control mice increased 3.1% compared to baseline values over the 6 weeks of the study, whereas grip strength measured for the first time in space showed flight animals to be -7.8% decreased in strength compared to baseline values. Control mice in space exhibited, compared to ground-based controls, a smaller increase in DEXA-measured muscle mass (+3.9% vs +5.6% respectively) although the difference was not significant. All individual flight limb muscles analyzed (except for the EDL) weighed significantly less than their ground counterparts at the study end (range -4.4% to -28.4%). Treatment with myostatin antibody YN41 was able to prevent many of these space-induced muscle changes. YN41 was able to block the reduction in muscle grip strength caused by spaceflight and was able to significantly increase the weight of all muscles of flight mice (apart from the EDL). Muscles of YN41-treated flight mice weighed as much as muscles from Ground IgG mice, with the exception of the soleus, demonstrating the ability to prevent spaceflight-induced atrophy. Muscle gene expression analysis demonstrated significant effects of microgravity and myostatin inhibition on many genes. Gamt and Actc1 gene expression was modulated by microgravity and YN41 in opposing directions. Myostatin inhibition did not overcome the significant reduction of microgravity on femoral BMD nor did it increase femoral or vertebral BMD in ground control mice. In summary, myostatin inhibition may be an effective countermeasure to detrimental consequences of skeletal muscle under microgravity conditions.
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RESEARCH ARTICLE
Inhibition of myostatin prevents microgravity-
induced loss of skeletal muscle mass and
strength
Rosamund C. SmithID
1
*, Martin S. Cramer
1
, Pamela J. MitchellID
1
, Jonathan Lucchesi
1
,
Alicia M. Ortega
2
, Eric W. Livingston
3
, Darryl Ballard
1
, Ling Zhang
1
, Jeff Hanson
1
,
Kenneth Barton
4
, Shawn Berens
1
, Kelly M. Credille
1
, Ted A. Bateman
3
, Virginia
L. Ferguson
2,5
, Yanfei L. Ma
1
, Louis S. Stodieck
2
1Lilly Research Laboratories, Indianapolis, Indiana, United States of America, 2Dept. of Aerospace
Engineering Sciences, BioServe Space Technologies, University of Colorado, Boulder, Colorado, United
States of America, 3Dept. of Biomedical Engineering, University of North Carolina, Chapel Hill, North
Carolina, United States of America, 4TechShot, Inc., Greenville, Indiana, United States of America, 5Dept.
of Mechanical Engineering, University of Colorado, Boulder, Colorado, United States of America
*Nonie23@att.net
Abstract
The microgravity conditions of prolonged spaceflight are known to result in skeletal muscle
atrophy that leads to diminished functional performance. To assess if inhibition of the growth
factor myostatin has potential to reverse these effects, mice were treated with a myostatin
antibody while housed on the International Space Station. Grip strength of ground control
mice increased 3.1% compared to baseline values over the 6 weeks of the study, whereas
grip strength measured for the first time in space showed flight animals to be -7.8%
decreased in strength compared to baseline values. Control mice in space exhibited, com-
pared to ground-based controls, a smaller increase in DEXA-measured muscle mass
(+3.9% vs +5.6% respectively) although the difference was not significant. All individual
flight limb muscles analyzed (except for the EDL) weighed significantly less than their
ground counterparts at the study end (range -4.4% to -28.4%). Treatment with myostatin
antibody YN41 was able to prevent many of these space-induced muscle changes. YN41
was able to block the reduction in muscle grip strength caused by spaceflight and was able
to significantly increase the weight of all muscles of flight mice (apart from the EDL). Mus-
cles of YN41-treated flight mice weighed as much as muscles from Ground IgG mice, with
the exception of the soleus, demonstrating the ability to prevent spaceflight-induced atro-
phy. Muscle gene expression analysis demonstrated significant effects of microgravity and
myostatin inhibition on many genes. Gamt and Actc1 gene expression was modulated by
microgravity and YN41 in opposing directions. Myostatin inhibition did not overcome the sig-
nificant reduction of microgravity on femoral BMD nor did it increase femoral or vertebral
BMD in ground control mice. In summary, myostatin inhibition may be an effective counter-
measure to detrimental consequences of skeletal muscle under microgravity conditions.
PLOS ONE
PLOS ONE | https://doi.org/10.1371/journal.pone.0230818 April 21, 2020 1 / 23
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OPEN ACCESS
Citation: Smith RC, Cramer MS, Mitchell PJ,
Lucchesi J, Ortega AM, Livingston EW, et al.
(2020) Inhibition of myostatin prevents
microgravity-induced loss of skeletal muscle mass
and strength. PLoS ONE 15(4): e0230818. https://
doi.org/10.1371/journal.pone.0230818
Editor: Makoto Kanzaki, Tohoku University, JAPAN
Received: December 2, 2019
Accepted: March 9, 2020
Published: April 21, 2020
Copyright: ©2020 Smith et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: Funding for this work was provided in
part to VLF, TAB, and LSS by the Center for the
Advancement of Science in Space or CASIS (GA-
2016-239, T/O #003). CASIS purchased mice used
in the study and funded associated animal testing
services. We acknowledge NASA for providing
access to astronaut crew members who carried
out the flight portion and all resources associated
with transportation to and from, and use of, the
Introduction
The microgravity conditions of spaceflight are known to result in a rapid atrophy of skeletal
muscle and also a loss of underlying bone [1,2]. The decrease of muscle mass leads to weakness
and diminished functional capacity, particularly noticeable in astronauts upon return to grav-
ity condition [36]. Rodents have proven to be a good animal model for muscle loss in space.
Lalani et al. [7] showed that rats flown on the Space Shuttle (STS-90) for 17 days lost 5% of
body weight and muscle weights (quadriceps, gastrocnemius, biceps, tibialis) were 19–24%
lower in weight than their ground controls. In 9-week-old (i.e., skeletally immature) female
C57Bl/6 mice flown on the Space Shuttle (STS-108, -135) for ~13 days, both body weight and
muscle mass (soleus, gastrocnemius, plantaris) were diminished where atrophy was indicated
by decreased myofiber cross-sectional area as compared to ground controls [810]. Decreased
myofiber CSA of soleus and EDL muscles was observed in skeletally mature 19–20 week-old
male C57Bl/6 mice flown on the BION-M1 biosatellite for 30 days [11]. The femoral quadri-
ceps muscle group from these same mice also showed significant atrophy and myofibril degen-
eration [12]. Long-term exposure to microgravity conditions of 91 days aboard the
International Space Station (ISS) interestingly showed no effect on myofiber CSA of the EDL
but a pronounced 35% decrease in soleus CSA [13], confirming that the type I soleus appears
particularly prone to wasting. In mice flown on the 30 day Bion-M1 mission, both fore- and
hindlimb grip strength was significantly reduced [10]. While direct biomechanical force mea-
surements in murine muscle following microgravity exposure is limited, the tibialis anterior
and masseter muscle fibers showed a correlation of decreased function with increased atrophy
[14]. With regards to effects of space on murine bones, a number of reports have described
diminished strength, microarchitecture, and mechanical properties following exposure of
mice and rats to microgravity [1519].
Skeletal muscle atrophy not only occurs during the unloading conditions of spaceflight, but
is also associated with many disease conditions on Earth such as cancer cachexia, muscular
dystrophy, sporadic inclusion body myositis, as well as with advanced aging (i.e., sarcopenia)
[20]. Loss of muscle mass results in reduced muscle function and this can have a direct nega-
tive impact on functional performance such as the ability to carry out activities of daily living
[21]. Muscle wasting has also been associated with poor prognosis and mortality [21]. While a
treatment for spinal muscle atrophy was approved in 2016 for clinical use in the United States
[22], medications that treat or prevent muscle atrophy remain largely unavailable. Exercise
and/or physical therapy remain the only options to counteract this condition. Astronauts in
space perform resistance exercises several hours a day to combat muscle wasting and bone
loss, which is both inefficient and insufficient [4,23]. With future goals of manned spaceflight
to the moon and Mars, especially if exercise equipment is limited, the ability to carry out mis-
sions may be compromised. On Earth compliance is also an issue for the ill and elderly. Fortu-
nately, a number of experimental medicines to treat muscle atrophy are making their way
through clinical trials. Many of these therapies have targeted the growth factor myostatin (also
known as GDF8) or its signaling pathway (reviewed in Smith and Lin, 2013) [24]. Myostatin is
a highly conserved secreted protein belonging to the TGFβsuperfamily of growth factors.
Myostatin is a negative regulator of vertebrate skeletal muscle mass as demonstrated by the
increased muscle mass and function seen in animals lacking functional copies of this growth
factor [24]. Genetic myostatin deficiency is also associated with increased bone cross-sectional
area, bone density and strength [25]. LY2495655 is an anti-myostatin antibody that has been
shown to cause an increase in muscle mass and function in mice [26]. A Phase 2a clinical trial
in the weak elderly demonstrated that inhibiting myostatin with LY2495655 was able to not
only increase appendicular lean body mass by 0.43 kg over the course of the 24-week trial,
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International Space Station. Additional support was
provided by NASA for the development of the
anesthesia/recovery system and anesthesia
protocol used in the study (NNJ10GA25A).
Development and provision of the ISS Bone
Densitometer was supported by CASIS and NASA
contracts NNJ13GA01C and NNJ10GA35C to
Techshot, Inc. Eli Lilly and Company and TechShot
Inc. provided support in the form of salaries for
authors [RCS, MSC, PJM, JL, DB, LZ, JH, SB,
KMC, YLM, KB], but did not have any additional
role in the study design, data collection and
analysis, decision to publish, or preparation of the
manuscript. The specific roles of these authors are
articulated in the ‘author contributions’ section.
Competing interests: The employment of authors
[RCS, MSC, PJM, JL, DB, LZ, JH, SB, KMC, YLM,
KB] at Eli Lilly and Company and TechShot Inc.
does not alter our adherence to PLOS ONE policies
on sharing data and materials.
but also to significantly improve stair climbing time, chair rise with arms, and fast gait speed
[27].
The goal of the current study was to determine if myostatin inhibition could prevent the
long-term microgravity-induced muscle atrophy expected in mice kept on board the Interna-
tional Space Station. For the first time in spaceflight, and onboard the ISS, measures of skeletal
muscle function as well as lean mass were collected in vivo on orbit at both interim as well as
terminal timepoints. In addition, further characterization of the effect of muscle and bone
unloading that occured in space was conducted. The results confirm and extend the character-
ization of the loss of muscle and bone induced in mice by spaceflight. In addition, a myostatin
antibody was able to prevent the loss of both muscle mass and function observed in micrograv-
ity. Not only do these results provide foundational preclinical data to support potential thera-
peutic intervention during long-duration space missions, but they also provide positive results
of myostatin inhibition in a unique and useful global animal model of muscle wasting not pos-
sible on Earth.
Materials and methods
Antibodies
YN41 (also called LSN2478185) is an anti-myostatin (GDF8) mouse IgG1 antibody that was
derived from injecting mice with full length mature myostatin with properties as described in
Smith et al. (2015) [26]. Control antibodies were IgG1 antibodies with known antigen binding
generated within Eli Lilly and Company. Antibodies were kept in long-term storage at -80˚C,
thawed prior to use and diluted with 1×PBS pH 7.4 (Invitrogen, Gibco) and stored at 4˚C for
the duration of the study. Pharmacokinetic and dynamic analysis of YN41 in mice supports
weekly dosing at 10 mg/kg [26] However, in order to reduce astronaut time, dosing was per-
formed every two weeks at 20 mg/kg with similar coverage and response.
Care and use of laboratory animals
All animal studies were conducted in strict accordance with the American Association for Lab-
oratory Animal Care institutional guidelines. All in vivo experimental protocols were approved
by both the NASA flight Institutional Animal Care and Use Committee (IACUC) based at
NASA Ames Research Center (Moffett Field, CA) and at the NASA Kennedy Space Center
(Protocol CAS-15-01-Y1).
Mice and live phase operations
Female BALB/cAnNTac mice were acquired from Taconic Farms (Germantown, NY) and
shipped to Kennedy Space Center (KSC) Animal Care Facilities at 8 weeks of age. Mice were
assessed for specific pathogen free (SPF) compliance prior to shipment from Taconic, within a
week of arrival at NASA KSC and at 10 days before launch (L-10 days). After arrival at KSC,
mice were adapted to spaceflight cage conditions including wire-mesh floors, spring operated
Lixit drinking mechanism and NASA-provided nutrient food bar diet [28]. At 1 week prior to
launch mice were caged at double density of n = 10 mice/cage to adjust to launch housing con-
ditions. Fifty mice were selected for inclusion in the study and assigned to equal-sized groups:
Baseline, Ground IgG, Ground YN41, Flight IgG, Flight YN41. Body weights across these
groups, which were 12 weeks of age at launch, averaged 19.3–19.8 g with a standard deviation
of 0.7 g.
At L-4 to L-2 days, baseline measures were collected on all animals including forelimb grip
strength and body composition by DEXA. Baseline controls (n = 10) were sacrificed on L+1
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and tissue collection carried out as described. One day prior to launch, flight and ground con-
trol animals were injected subcutaneously (SQ) with control IgG (Flight IgG) or YN41 myosta-
tin antibodies (Flight YN41) at 20 mg/kg in a total dosing volume of 200 μl prior to loading
into the transportation habitat. YN41 treated mice were loaded into one side of the Trans-
porter housing system [29] and IgG mice into the other side (n = 10 mice/side); ground con-
trols were similarly loaded into a NASA mouse Transporter. The Transporter habitat was
loaded into the Space-X Dragon capsule and launched into low Earth orbit on the CRS-8 mis-
sion on April 8
th
, 2016. The capsule berthed with the ISS at 41 hours post-launch, and approxi-
mately 90 hours post launch mice were transferred to 2 ISS Habitats with each having 2
compartments (i.e., sides) that housed 2 controls and 3 treated animals and vice-a-versa. Mice
groups were further dosed with YN41 myostatin antibodies or control IgG antibodies at 20
mg/kg in 200 μl at 2 and 4 weeks post-launch.
The ground control groups (Ground IgG, Ground YN41) were treated as identically as pos-
sible to flight animals, including housing in the same Transporter and Habitat housing sys-
tems. Ground controls were offset from flight animals by 3 days (L+3) to enable exposure to
matched environmental conditions (i.e., temperature, humidity and carbon dioxide levels).
At 4 weeks post-launch, forelimb grip strength and body composition via Dual Energy X-
ray Absorptiometry (DEXA) were collected over a period of 3 days (note: this extended dura-
tion is due to limited astronaut crew time on the ISS). At 6 weeks post-launch and 2 days prior
to termination, grip strength was again assessed. Body composition measures via DEXA were
collected just prior to termination. For termination, both ground control and flight mice were
anesthetized and subjected to cardiac puncture followed by cervical dislocation.
Following termination, the skin was removed from the right hindlimb and the entire limb
detached at the pelvis and immersion fixed in 10% neutral buffered formalin and stored at
room temperature. The remaining whole carcass was stored at -80˚C (ground) or -95˚C
(flight) until time of dissection. Flight samples were returned to Earth on SpX-9 on August 26,
2016. All additional tissues and organs were collected and processed from both control and
flight frozen carcasses as outlined below.
Grip strength measures
Forelimb grip strength measures [30] were acquired with a digital Grip Strength Meter
(Columbus Instruments Model 0167-004L). Identical instruments were used to assess flight
mice on the ISS and ground controls. At each time point (0, 2, 4, and 6 weeks post launch),
four-limb measurements were acquired in the compression peak (C PEAK) mode. A total of 4
readings were recorded from each mouse with the lowest and highest readings excluded and
the remaining values averaged.
In vivo body composition measures
Body composition was measured by DEXA using a Lunar PIXImus (GE Lunar Corp, WI) or a
derivative thereof modified for spaceflight and known as the ISS Bone Densitometer (Tech-
shot, Greenville, IN, USA). Mice were anesthetized for pre-flight data collection through iso-
flurane inhalation (baseline and pre-flight animals). During the mission, two body
composition measurements were obtained from flight and ground control animals. For the
first intermediate time point (4 weeks after launch) and to accommodate the need to anesthe-
tize mice on the ISS, mice were injected IP with ketamine/xylazine (80/15 mg/kg, flight and
ground controls). Upon completion of DEXA measurements, mice were administered atipa-
mezole IP (1 mg/kg), placed individually into a warming device (36˚C) and allowed to fully
recover before being returned to their Habitats. At termination, mice were anesthetized with
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ketamine/xylazine/acepromazine (120/15/3 mg/kg) and DEXA measures collected prior to
cardiac puncture.
DEXA-based bone mineral density (BMD) and lean mass values were based on a region of
interest (ROI) centered on the hindlimb and hip regions. The ROI was established between the
ankle joint and the iliac crest (6
th
lumbar vertebra) and between the outermost regions of the left
and right hindlimbs (Fig 1C). For BMD measurements the correction factor between the ISS
Bone Densitometer and the ground-based Lunar PIXImus was determined to be 1.000 ±0.009 so
the data was uncorrected. Lean body mass was also derived from DEXA data, in which case the
correction factor from the ISS Bone Densitometer to the Lunar PIXImus was determined to be
0.928 ±0.010, which was applied to the lean body mass determined during space flight.
Body weight and tissue collection
Carcasses were carefully thawed, the left hindlimb was immediately removed, and the gastroc-
nemius, quadriceps, tibialis anterior, soleus, plantaris and extensor digitorum longus (EDL)
muscles were collected, weighed and placed in RNALater. Bones were collected and cleaned of
non-bony tissue, and stored in 70% ethanol at room temperature for subsequent analyses.
Heart and brain weights were also collected. Body weights of all mice did not include the skin
of the right hindlimb.
Fig 1. Effect of myostatin inhibition on muscle function and lean muscle mass under conditions of microgravity. Grip
strength (A) and lean muscle mass (B) were measured in Ground IgG, Ground YN41, Flight IgG and Flight YN41 groups at
launch and weeks 4 and 6 post-launch, expressed as percent change from the same groups measured at baseline, with all
baseline measurements having been made on the ground using Lunar PIXImus. (C) Representative mouse image from the
DEXA densitometer showing the region of interest (ROI) of the hindlimb outlined in black used in panel B. (D) Carcass
weights of all groups. Significance (p <0.05 , 0.001 ) for comparison to respective control IgG group or to Ground IgG
group (p <0.05 #, p <0.001 ##) is noted.
https://doi.org/10.1371/journal.pone.0230818.g001
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Muscle and bone histology
After fixation and return to Earth, the lower hindlimb muscle bundle (i.e., containing gastroc-
nemius, soleus and plantaris) was dissected from the right hindlimb and bisected to obtain
cross sections of the muscle fibers. The bundle was trimmed at three levels: proximal, mid-
muscle bundle and distal. The trimmed muscle samples were routinely processed and embed-
ded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin and a histochemical
stain, Chandler’s reticulum, to highlight the muscle fiber basement membrane for image anal-
ysis. The intact pelvic limbs were routinely decalcified. After decalcification the femur was
carefully disarticulated from the coxofemoral joint and was transected proximal to the femo-
ral-tibial joint, routinely processed and paraffin embedded with a longitudinal orientation to
allow evaluation of the femoral head and articular cartilage. The tibia was transected below the
femoral-tibial joint in order to isolate the joint. The joint was bisected along the frontal plane,
routinely processed and the cut surface embedded in paraffin so that the exposed center of the
joint was sectioned to allow evaluation of articular cartilage and other joint tissues.
The bone sections were stained with toluidine blue. The stained slides for all muscle and
bone samples were examined by a pathologist. The reticulum-stained sections of the gastroc-
nemius mid-bundle were digitized for image analysis using an Aperio Scan Scope AT2 (Leica
Biosystems).
Muscle fiber quantitation
Muscle fiber cross-sectional area was measured in the scanned images using the Halo software
package MuscleFiber module (MuscleFiber v2.2.1, HALO v2.0 1145.31) (Indica Labs). Briefly,
each entire reticulum-stained gastrocnemius bundle section was outlined in the Halo software.
Histology artifacts (section tears, wrinkles), anatomic structures that interfered with muscle
fiber recognition (tendons, oblique fibers) and poorly detected muscle fibers were removed
manually from analysis using the software exclusion tool. The MuscleFiber module identified
and measured all of the remaining fibers in the bundle, reporting the total fiber count and
average fiber area. The average fiber area was used for comparison across groups. One entire
mid-bundle section was quantified per mouse averaging 8186 fibers per section (ranging from
3828–12140 fibers per section).
Ex vivo bone measures and biomechanics
Femora and L5 vertebrae were analyzed by quantitative microcomputed tomography
(microCT) at two length scales using, first, a LaTheta LTC-100 CT scanner (Aloka; 100 μm
voxels) [31]. Briefly, a scan of each femur was performed at both 0.4 and 4.4 mm from the end
of the growth plate for analysis of the distal metaphysis and midshaft, respectively. The femur
metaphysis contains both trabecular and cortical bone whereas the midshaft femur contains
only cortical bone. Aloka software (SYS-C320 version 1.5) was used to assess BMC and bone
area for the entire cross-section of both trabecular and cortical bone as in Smith et al. (2012)
[31]; area BMD was calculated as BMC normalized to bone volumetric area. The left, distal
femur was next evaluated for cortical and trabecular bone microarchitecture [32] (μCT80;
Scanco Medical AG, Bruttisellen, Switzerland; 70 kVp, isotropic voxel size of 10 μm). Volumes
analyzed were located at the distal femur metaphysis, starting 0.5 mm proximal to the epiphy-
seal line and extending 1 mm proximally. Trabecular and cortical bone were each indepen-
dently segmented and analyzed using SCANCO evaluation software according to established
guidelines [33]. Measured trabecular bone parameters included bone volume fraction (BV/
TV), trabecular bone number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.
Sp), and trabecular volumetric bone mineral density (vBMD). Cortical bone measures
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included bone area and total area (B.Ar and T.Ar; i.e., the mean cross-sectional areas of the
analyzed volume), cortical thickness (Ct.Th), and cortical bone vBMD which describe the
amount of bone contained in the region that was evaluated. Also included are cortical porosity
(Ct.Po), which indicates subcortical resorption and is a contributor to bone strength, and max-
imum and minimum moments of inertia (I
max
and I
min
), which contribute to bone structural
integrity and strength.
Biomechanical properties of the femur were evaluated using three-point bending as previ-
ously described [34]. Briefly, femur length was measured via calipers (Mitutoyo, Kanagawa,
Japan). Mechanical testing was performed in a 37 ˚C saline bath (MTS model 1/S, 100 N load
cell; analyzed using TestWorks 4 software; MTS Corp., Minneapolis, MN) with testing support
span width of 8 mm and crosshead speed of 0.17 mm/s.
Gene expression studies
Total RNA was isolated from muscle tissue using mirVana kit from Invitrogen. Briefly, tissues
were homogenized in kit lysis buffer with Matrix D beads in Fast Prep-24 Instrument for two
60 second runs at Speed 6. The homogenate was processed according to kit instructions and
components using phenol:chloroform extraction and spin column purification. RNA integrity
number (RIN) and concentration was determined using Nanodrop and Agilent Bioanalyzer
using Agilent Nano kit. Total RNA samples with a RIN value below 6.0 were not processed fur-
ther. The cDNA for each RNA sample was prepared using the High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems) according to kit instructions. For quadriceps, gastroc-
nemius, and tibialis anterior, cDNA reactions contained 5 μg total RNA per 100 μl, while for
soleus, cDNA reactions contained 2 μg total RNA per 40 μl reaction. The cDNA was diluted
1:10 in water for quantitative PCR analysis. QPCR was performed with two technical replicates
using TaqMan Fast Advanced Master Mix (Applied Biosystems), validated gene expression
assays (Invitrogen), and eukaryotic 18s rRNA (Applied Biosystems) on the QuantStudio 7 Flex
Instrument. Results are based on DeltaDelta CT calculations from gene of interest CT and 18s
CT values.
Statistical analyses
Measures collected at multiple time points during the study including grip strength and those
from DEXA (lean mass and BMD) were evaluated by a repeated measures ANOVA, and con-
firmed using MANOVA to account for variations in sphericity, while considering factors of
spaceflight, treatment, and time (0, 4, and 6 weeks post launch). Endpoint measures were ana-
lyzed statistically using Student’s t-test to compare baseline to controls, where appropriate,
and using two-way Analysis of Variance (ANOVA) tested for group-wise comparisons for
effects of spaceflight (ground vs flight) and treatment (IgG and YN41). Post-hoc analyses were
performed using Dunnett’s test (micro-computed tomography) or Student’s paired ttest. Sta-
tistical outliers were removed from QPCR data (defined as those beyonds 1.5 x Interquartile
range from the first or third quartile) but not from other datasets. For all tests, a Pvalue
of <0.05 was considered statistically significant. Error bars on all graphs show standard error
(s.e.m.) from the mean.
Results
Visual inspection of mice upon transfer and inspection of video footage of the mice in their
on-orbit habitats after transfer, and daily throughout the mission, revealed that the mice
adapted well to their habitats and appeared healthy. All flight and ground control mice sur-
vived the live phase portion of the experiment and appeared healthy at termination.
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Effects of myostatin inhibition on muscle function under conditions of
microgravity
The results of the grip strength measures at interim and final timepoints showed significant
main effects of both spaceflight and myostatin inhibition (p <0.0001) and that grip strength
was influenced by time following launch (p <0.001). Statistically significant interactions were
observed between time and spaceflight and time and myostatin treatment. Group-wise com-
parisons showed that grip strength in the Ground IgG control levels differed from baseline lev-
els by -3.7% and +3.1%, respectively, at 4 and 6 weeks, respectively (Fig 1A). In comparison,
administration of the myostatin antibody YN41 significantly increased grip strength by
+14.6% over 4 weeks and +12.9% over 6 weeks. The increased grip strength results are consis-
tent with previous reports using this antibody [26]. Muscle function significantly declined
from baseline levels in Flight IgG group (-19.3% at week 4 and -7.8% at week 6), and at both
timepoints the Flight IgG grip strength measurements were significantly lower than Ground
IgG levels (p<0.05) at equivalent timepoints. These results demonstrated that microgravity
conditions resulted in a decrease in muscle function as early as 4 weeks into flight. Grip
strength increased at week 6 compared to week 4 in both flight animals and ground IgG con-
trols, which might follow growth and maturation from the start of the flight experiment at 12
weeks of age. YN41 treatment of flight animals prevented the microgravity induced loss in
muscle function. Indeed, the percentage change in grip strength from week 0 to 6-week of
Flight YN41 animals was not significantly different to that of Ground IgG animals. Moreover,
the percentage change in grip strength for YN41 Flight animals of 0.6% versus -7.8% for Flight
IgG animals from week 0 to week 6 was similar in magnitude to the effect seen in the Ground
YN41 group compared to its IgG group (12.9% vs 3.1%).
Effects of microgravity and myostatin inhibition on skeletal muscle mass
and body weight
To explore further the underlying physiology behind the effects of microgravity and myostatin
inhibition on muscle function, in vivo body composition was analyzed using a DEXA densi-
tometer at baseline, interim, and terminal timepoints. The results of this analysis are focused
on lean mass in the hindlimb region (Fig 1B and 1C). Lean mass in ground control animals
increased over the course of the experiment, likely due to normal growth during the study
period, with main effects of both spaceflight (p = 0.066) and myostatin inhibition (p <0.05).
Lean mass changed with time following launch (p <0.0001), and time was interactive with
both spaceflight (p <0.01) and myostatin inhibition (p <0.01). Lean mass gains in Flight IgG
animals were less than in ground IgG animals over the 6 week experiment (1.0 vs 3.5% gain at
week 4, for Flight and Ground IgG groups, respectively; and 3.9% vs 5.6% gain at week 6),
although the effect was not significant. In ground animals, YN41 treatment significantly
increased lean mass at both 4 and 6 weeks as compared to untreated controls, and lean mass
was also increased for treated flight animals as compared to untreated flight controls (3.6% vs
1.0% at week 4, for YN41 and IgG flight mice, respectively; and 7.9% vs 3.9% at week 6).
To determine if the observed changes in lean mass influenced body mass, the carcass
weights (minus skin and the right leg) of all animals were assessed. The overall body weight in
IgG control groups tended to decrease with microgravity exposure but the effect was not sig-
nificant (Fig 1D). YN41-treated animals, however, were significantly heavier than their IgG
controls, both in flight and on the ground, which was consistent with increases in lean mass
observed using DEXA.
Individual muscles were dissected and weighed. All muscles evaluated from the ground IgG
group increased in weight (ranging from 7.5–19.5% over baseline values) over the 6-week
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course of the live phase experiment, and as expected of 12-week old growing mice (Table 1). In
contrast, both fore-limb and hindlimb muscles (except for the EDL) from Flight IgG mice did not
gain as much as the ground controls over the course of the experiment. The flight gastrocnemius
did not gain any weight compared to the baseline group (Fig 2A) and the flight soleus decreased
by -14.4% as compared to baseline values (Fig 2D). The quadriceps (Fig 2B) and tibialis anterior
(TA) muscles from Flight IgG mice were also significantly lighter compared to those from
Ground IgG mice. Comparably, the EDL appeared not to atrophy from spaceflight, in line with
muscle masses [13] and gene expression changes observed in prior studies of spaceflight flown
mice [13]. Two-way ANOVA showed a main effect of microgravity exposure affected all but the
triceps brachii and EDL muscles. Overall, our results support the reduced hindlimb lean mass and
muscle strength observed in Flight IgG animals as compared to the ground IgG group.
Myostatin antibody treatment of ground control mice significantly increased the weight
of 6 of the 7 individual muscles isolated (Table 1,Fig 2A–2C) from 12.9–31.6% depending on
the individual muscle. The soleus, a type I slow oxidative muscle, showed an increased in
weight with YN41 treatment, but the effect was not significant (Fig 2D). All other muscles
(except the EDL) isolated from Flight YN41 mice were significantly heavier than the Flight
IgG controls (Fig 2A–2C), ranging from 10.5–28.0% heavier depending on the individual mus-
cle, and demonstrating that myostatin inhibition can occur under conditions of microgravity.
Notably, 5 of these muscles were also significantly heavier than their Ground IgG counterparts
demonstrating that myostatin inhibition was able to prevent all the muscle atrophy induced by
conditions of microgravity. It is interesting to compare the degree of effect of myostatin inhibi-
tion on individual muscles on Earth as compared to on the ISS. A number of the muscles such
as the gastrocnemius, quadriceps and triceps responded equally well in the presence or absence
of gravity. The TA and soleus appeared to respond better under microgravity conditions as
compared to equivalent ground controls, whereas the EDL and plantaris responded to a lesser
degree than their ground control muscles (Table 1).
Table 1. Effect of myostatin inhibition on individual muscle weights under conditions of microgravity.
Muscle Weight
Baseline Ground IgG Ground YN41 Flight IgG Flight YN41
mg mg % Change
from
Baseline
mg % Change
from Ground
IgG
mg % Change
from Ground
IgG
mg % Change
from Ground
IgG
% Change
from Flight
IgG
2 way ANOVA
MI,SF,Int
Triceps brachii 73.3±1.8 81.9
±2.2
11.8±3.0107.8
±2.3
31.6±2.7�� 78.3
±3.9
-4.4±4.8 100.3
±2.5
22.4±3.1�� 28.0±3.4‡‡‡,,NS
Gastrocnemius 83.6±1.7 95.5
±3.3
14.3±4.0112.4
±2.0
17.7±2.0�� 83.6
±2.0
-12.5±2.1100.3
±3.7
4.9±3.9 20±4.4‡‡‡,‡‡‡,‡‡
Quadriceps 141.0
±4.9
158.8
±3.9
12.7±2.8196.0
±2.5
23.4±1.5�� 144.9
±3.4
-8.8±2.1177.7
±5.1
11.9±3.222.6±3.5�� ‡‡‡,‡‡,NS
Tibialis anterior
(TA)
44.1±1.6 51.6
±1.1
16.9±2.658.2
±1.8
12.9±3.346.7
±1.1
-9.5±2.159.2
±0.8
14.8±1.5�� 26.9±1.7‡‡‡,‡‡‡,††
Extensor digitalis
longissimus (EDL)
8.3±0.2 8.9
±0.2
7.5±2.6 10.8
±0.3
21.8±3.7�� 9.0
±0.3
1.5±3.6 10.0
±0.3
12.2±3.410.5±3.3 ‡‡‡,NS,NS
Plantaris 10.1±0.4 11.4
±0.4
12.9±3.814.2
±0.3
24.9±2.8�� 10.7
±0.2
-5.9±1.7 12.7
±0.5
11.7±4.6 18.7±4.9‡‡‡,,NS
Soleus 5.6±0.2 6.6
±0.3
19.5±4.76.9
±0.2
3.6±2.5 4.8
±0.2
-28.4±2.2�� 5.3
±0.2
-20.4±2.3�� 11.2±3.2,‡‡‡,NS
Two-way ANOVA evaluated for main effects of myostatin inhibitor treatment (MI: IgG and YN41) and spaceflight (SF: ground vs flight), and interactions (Int) between
SF and Tmt; p-values are reported for † = p <0.10, †† = p <0.05, ‡ p <0.01, ‡‡ p <0.001, ‡‡‡ p <0.0001, and NS indicates no significance. For % change over
Baseline, Ground IgG or Flight IgG comparisons: indicates significance p <0.05; �� indicates significance p <0.001; data are presented as mean ±s.e.m.
https://doi.org/10.1371/journal.pone.0230818.t001
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Heart and brain weights were collected from all five treatment groups (Fig 2E and 2F,
respectively). Heart weights were significantly lighter for the Flight IgG group compared to the
Ground IgG group; two-way ANOVA also showed a main effect of spaceflight but not treat-
ment for heart mass. YN41 treatment overcame this reduction. YN41 treatment did not affect
heart weights of ground controls. Brain weights did not change over the course of the live
phase of the experiment and neither microgravity and/or myostatin inhibition affected this
outcome.
Effect of microgravity and myostatin inhibition on muscle
histomorphology and fiber size
To explore the effects of microgravity and myostatin inhibition on underlying hindlimb mor-
phology, sections of hindlimb muscle bundles and femoral heads from flight and ground con-
trol mice were examined histologically. It has been suggested that cartilage integrity may be
compromised by long-term spaceflight [35]. To this end, femoral heads from mice exposed to
microgravity were examined but showed no inflammatory or degenerative changes regardless
of drug treatment (Fig 3Ai-iv). Muscles from ground and flight mice without YN41 treatment
also appeared histologically normal (Fig 3Bi-iv).
Since microgravity had been shown to reduce individual muscle masses in the hindlimbs of
flight mice, it was of interest to determine if this was due to an atrophy of existing myofibers.
Myofiber size of muscles in the hindlimb lower muscle bundle was assessed using an image
analysis algorithm, where image analysis tools recognize and outline myofibers for measure-
ment of cross-sectional area (Fig 3D). Although there was a decrease in CSA in muscles from
Fig 2. Effect of myostatin inhibition on muscle, brain and heart weights under conditionsof microgravity. Individual weights of (A) gastrocnemius, (B) quadriceps,
(C) plantaris, (D) soleus, (E) heart, (F) brain for Ground IgG, Ground YN41, Flight IgG and Flight YN41 groups at weeks 4 and 6 post-launch expressed as % change
from the baseline group (A-D) or absolute weight (E-F). ,Indicates significance vs. its respective IgG control; #, ## indicates significance vs. ground IgG. p <0.05,
p<0.001.
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Fig 3. Effect of myostatin inhibition and microgravity on femoral head and muscle histology and myofiber size.
A). No degenerative or inflammatory changes were microscopically observed in observed in longitudinal sections of
the femoral head near the ligament of the head of the femur (A) or in transverse sections of the lower hind limb
muscles (B) in control or YN41 treated mice housed on the ground or in flight. i) Ground IgG, ii) Ground YN41, iii)
Flight IgG, iv) Flight YN41. High magnification inset in B is of the soleus muscle (centrally located). (C). Mean cross-
sectional areas of lower limb hindlimb muscle fibers from ground and flight groups, where two-way ANOVA showed
main effects of both myostatin inhibition (p <0.0001) and spaceflight (p <0.05). p<0.001 vs their respective IgG
controls. D). Representative image of a lower limb muscle section (top) stained to highlight muscle fibers and its
corresponding image analysis mask (bottom) used as part of the image analysis process.
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Myostatin inhibition prevents microgravity-induced loss of skeletal muscle mass and strength
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Flight IgG compared to Ground IgG (Fig 3C), the decrease was not significant. YN41 treat-
ment, however, caused a hypertrophy of myofibers for both control and flight mice as shown
by the significant increase in myofiber CSA.
Effect of microgravity and myostatin inhibition on muscle gene expression
To investigate the molecular effects of microgravity and myostatin inhibition on skeletal mus-
cle of ground and flight mice, gene expression was analyzed using QPCR. Three classes of
genes were selected for analysis: i) genes previously described to be modulated by micrograv-
ity, ii) genes belonging to the myostatin pathway, iii) genes known to be modulated by myosta-
tin inhibition (previously published or from our own unpublished observations). Some genes
fit into more than one category. The genes, their descriptions and the primer probe kits used
for these analyses are listed in S1 Table. The expression results for these 28 genes in the gas-
trocnemius, quadriceps, soleus and tibialis anterior muscles are tabulated in S2 Table. It is to
be noted that these genes often had very different levels of expression between the 4 muscles
examined in control ground-dwelling mice. For example, Mybph, Actc and Resn were
expressed more highly in the fast-twitch quadriceps muscle but virtually undetectable in the
slow-twitch soleus. Conversely, Fst and Cyr61 and Pax7 were expressed at a higher level in the
soleus than the quadriceps. Even within fast twitch muscles, gene expression levels varied in
untreated ground control mice. For example, Cidec1 was highly expressed in the quadriceps
but at 10-fold less levels in the gastrocnemius and TA. The reasons for these varying gene
expression levels between individual muscles is unclear. It was of interest to determine if
microgravity had modulated gene expression in skeletal muscle concomitant with inducing
reductions in muscle mass. Alpha cardiac actin (Actc1) was significantly affected by micro-
gravity in 3 out of the 4 muscles examined (Fig 4A). A decrease in gene expression in Flight
IgG animals compared to Ground IgG animals was seen in the gastrocnemius and TA muscles
whereas an increase was seen in the soleus. Guanidinoacetate methyltransferase (Gamt), an N-
methyltransferase that catalyzes synthesis of creatine, was significantly increased by spaceflight
in the quadriceps (Fig 4C), whereas Myosin binding protein H (Mybph), was significantly
decreased by microgravity in the gastrocnemius (Fig 4B). A decrease in expression of resistin
(Retn) in the TA in response to spaceflight was observed (Fig 4D). The next set of 17 genes
examined had previously been described as being modulated by spaceflight [11,13,36]. A sig-
nificant decrease in Frzd9 expression in the gastrocnemius in flight animals was observed,
reproducing the result of Allen et al. [36], with an increase seen in the TA (S2 Table). Kcnma1
was significantly decreased (-1.72 fold) in the soleus in flight animals in accordance with the
result of Gambara, et al. [11]. Trim63 (MurF1) showed a significant increase in the soleus, in
accordance with data from Sandona, et al. [13]. Pitx2, a gene shown to be increased by micro-
gravity in the gastrocnemius [36], showed a trend for an increase in flight IgG animals com-
pared to ground controls and a significant increase (+1.54 fold) in flight animals in the
quadriceps. Ppargc1a showed a significant decrease in flight in the gastrocnemius but did not
reproduce the previously decrease in flight in the soleus [11]. Rather than a decrease seen in
flight in the gastrocnemius of Slc38a2 expression as reported previously [36], a significant
increase in this gene in flight animals was observed (S2 Table). No significant changes were
seen in muscles from flight IgG mice compared to ground IgG mice in the expression of Cfd,
Cidec, Cyr61 (Fig 4F), Dnajb1, Fasn, Foxo1, Fst (Fig 4E), Fstl1, Id1, Myf6 or Rbp4 (S2 Table),
all genes previously reported to be modulated by microgravity.
A number of significant changes in gene expression were induced by myostatin inhibition,
particularly in the fast twitch muscles (i.e., gastrocnemius, quadriceps and tibialis anterior) on
which YN41 had a pronounced effect on muscle mass. Effects on gene expression were less
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Myostatin inhibition prevents microgravity-induced loss of skeletal muscle mass and strength
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Myostatin inhibition prevents microgravity-induced loss of skeletal muscle mass and strength
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pronounced in the soleus, in keeping with the lesser effect of myostatin inhibition on the
weight of this muscle. In general, effects of myostatin inhibition on gene expression were simi-
lar in flight as in ground animals. Actc1 and Mybph both showed increases in expression in
the gastrocnemius and TA in both ground and flight animals with YN41 treatment (Fig 4A
and 4B). Previously Latres et al. [37] described that treatment of mice with a myostatin anti-
body increased expression of Actc1 and Mybph in the TA. Interestingly, for both Actc1 and
Mybph, myostatin inhibition modulated expression in the opposite direction to the effect of
microgravity. Notably, myostatin inhibition in the gastrocnemius was able to overcome the
effects of microgravity and restore gene expression levels to at least those of Ground IgG con-
trols (Fig 4A). Gamt was also significantly increased in the gastrocnemius and quadriceps of
both ground and flight animals with myostatin inhibition although in the quadriceps micro-
gravity also increased Gamt expression. For Trim63, myostatin inhibition in the soleus of flight
animals was shown to significantly decrease expression, whereas microgravity modulated the
gene in the opposing direction (S2 Table). A number of genes eg Fst, Cyr61, Itgb5, Resn and
Zymd17 were significantly decreased by YN41 treatment in both Ground and Flight animals
and across multiple muscles (Fig 4D–4H). Others have previously reported a decrease of
Zymd17 in the TA of myostatin antibody-treated mice [37]. The only effect of microgravity on
these genes was in a decrease in the TA of Resn in flight animals and an increase in the soleus
of Zymd17. However, since the expression of these 2 genes was very low in these specific mus-
cles, the significance of this finding is unclear. Other genes that were influenced by YN41 treat-
ment, either on the ground or in flight, in at least one muscle were Acrv2b, Dnajb1, Fasn,
Fstl1, Frzd9, Igfbp5, Pax7, Pitx2, Ppargc1a and Rbp4 (S2 Table). Myostatin gene expression
was shown to be increased by YN41 treatment in the gastrocnemius, perhaps as a compensa-
tory mechanism for myostatin protein sequestration (S2 Table).
Effect of microgravity and myostatin inhibition on bone
The effects of microgravity and myostatin inhibition on bone health were evaluated via DEXA
(i.e., for bone mineral density), micro-computed tomography (e.g., for areal bone mineral den-
sity and microarchitectural assessment), and mechanical testing (e.g., for strength). Interim (4
and 6 weeks post launch) and terminal in vivo DEXA bone densitometer readings were taken
for flight and ground mice and the data are shown in Fig 5A. Microgravity caused a significant
decrease in areal bone mineral density (BMD) in the region of the hindlimb (region shown in
Fig 1C) in Flight IgG animals compared to their Ground IgG counterparts (Fig 5A). The
decrease in areal BMD in flight mice was apparent after 4 weeks onboard the ISS, where the
group-wise effect of spaceflight was significant (p <0.01). Myostatin inhibition had no signifi-
cant effect on BMD from DEXA either on the ground or in flight; however, time following
launch was highly significant (p <0.0001) and time was interactive with spaceflight
(p <0.0001) but not myostatin inhibition treatment. To examine the effect on bone density in
more detail, femurs and vertebrae were examined for bone-specific areal BMD by computed
tomography (Table 2) and then for microarchitectural assessment at the distal femur. As seen
with in vivo DEXA bone measures, ex vivo CT confirmed that conditions of microgravity
resulted in a significant decrease in areal BMD in the femur but not in the vertebrae. Mid- and
distal femoral areal BMD were -10.76% and -7.10% lower in Flight IgG animals compared to
Ground IgGs, respectively (Table 2,Fig 5B and 5C). However, lumbar vertebral areal BMD
Fig 4. Effect of microgravity and myostatin inhibition on skeletal muscle gene expression. Gene expression profiles of 8 genes in gastrocnemius (G),
quadriceps (Q), soleus (S) and tibialis anterior (T) muscles of flight and ground animals. Significance at p<0.05, p<0.001 relative to the respective control
groups (,��), to the Flight IgG group (#,##) and to the Ground IgG group ($,$ $).
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was unaffected by conditions of microgravity (Table 2). As observed in BMD measurements
from DEXA, myostatin inhibition had no significant effect on femoral or vertebral BMD as
measured ex vivo (Table 2,Fig 5B and 5C).
Fig 5. Effect of microgravity and myostatin inhibition on bone density. A. Areal bone mineral density measured in vivo by DEXA in Ground IgG, Ground YN41,
Flight IgG and Flight YN41 groups at weeks 4 and 6 post-launch expressed as % change from the same groups measured at week 0. (), refers to significance relative to
its respective control at each time point. All p values <0.05. BMD of distal (B) and mid (C) femurs as measured by ex vivo CT. denotes significance to Ground IgG,
p<0.05. Two-way ANOVA revealed significant main effects of spaceflight, but no significant effect of myostatin inhibition, for areal BMD at both 4 and 6 weeks post-
launch (p <0.01 and p <0.0001, respectively) and BMD of distal and mid-femurs (p <0.0001).
https://doi.org/10.1371/journal.pone.0230818.g005
Table 2. Age and microgravity, but not myostatin inhibition, influence vBMD in the femur but not the vertebrae. Age and microgravity, but not myostatin inhibition,
also influence both trabecular and cortical bone microarchitecture at the distal femur.
Baseline Ground IgG Ground YN41 Flight IgG Flight YN41 2 way ANOVA MI,SF
Bone-Specific Areal BMD Measures (all in mg/cm
2
)
Mid- Femur 619.6 ±8.9 † 582.8 ±12.8
A
588.0 ±11.2
A
520.1 ±10.4
B
533.5 ±11.0
B
‡‡‡, N.S.
Distal Femur 653.1 ±6.0 647.0 ±8.7
A
660.3 ±7.3
A
601.1 ±7.8
B
608.1 ±7.9
B
‡‡‡, N.S.
Lumbar Vertebrae 324.9 ±9.4 343.5 ±10.9 363.3 ±10.8 356.3 ±12.7 356.6 ±13.0 N.S., N.S.
Trabecular Bone Microarchitecture Measures
Bone Volume Fraction, BV/TV (%) 12.4 ±0.9 14.6 ±1.0 13. 6 ±1.0 11.2 ±1.2 12.1 ±0.5 ‡, N.S.
Trabecular Number, Tb.N (1/μm) 3.19 ±0.17 3.61 ±0.17 3.42 ±0.16 2.97 ±0.22 3.19 ±0.10 ††, N.S.
Trabecular Thickness, Tb.Th (μm) 37.3 ±1.0 39.5 ±1.2 38.8 ±1.1 36.0 ±1.3 37.1 ±0.8 ††, N.S.
Trabecular Spacing, Tb.Sp (μm) 286 ±21 245 ±17
A
260 ±15
AB
319 ±29
B
279 ±9
AB
††, N.S.
Tb.vBMD 153.5 ±9.0 183.1 ±10.9
A
169.5 ±11.0
AB
140.6 ±13.0
B
152.4 ±6.3
AB
‡, N.S.
Cortical Bone Microarchitecture Measures
Bone Area, BA (mm
2
) 0.89 ±0.01 † 0.94 ±0.02
AB
0.99 ±0.02
A
0.88 ±0.02
B
0.88 ±0.02
B
‡‡‡, N.S.
Total Area, TA (mm
2
) 1.09 ±0.01 1.11 ±0.02
AB
1.17 ±0.02
A
1.06 ±0.02
B
1.05 ±0.03
B
‡‡, N.S.
Cortical Thickness, Ct.Th (μm) 134.35 ±1.85 ‡ 151.10 ±1.84
AB
155.82 ±1.67
A
148.96 ±2.02
B
147.26 ±1.61
B
‡, N.S.
Cortical Porosity, Ct.Po (%) 18.1 ±0.5 ‡ 15.3 ±0.3
AB
14.9 ±0.4
B
16.5 ±0.5
A
15.9 ±0.4
AB
‡, N.S.
I
max
(mm
4
) 0.43 ±0.01 § 0.48 ±0.01
AB
0.52 ±0.01
A
0.45 ±0.01
B
0.44 ±0.02
B
‡‡, N.S.
I
min
(mm
4
) 0.20 ±0.00 § 0.22 ±0.01 0.23 ±0.01 0.21 ±0.00 0.21 ±0.01 ††, N.S.
Ct.vBMD (mg HA/ccm) 892.0 ±5.5 ‡ 944.6 ±2.9 948.8 ±8.7 935.3 ±5.2 929.8 ±6.8 ††, N.S.
Two-way ANOVA evaluated for main effects of myostatin inhibitor treatment (MI: IgG and YN41) and spaceflight (SF: ground vs flight); no interactions were observed.
P-values are reported for †† = p <0.05, ‡ p <0.01, ‡‡ p <0.001, ‡‡‡ p <0.0001, and NS indicates no significance.
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Data are reported as mean ±s.e.m. Baselines were compared to ground IgG groups using a
Student’s t-test to evaluate for influence of age during the flight experiment. All differences are
noted as † for p <0.05 § for p <0.01, ‡ for p <0.001. Two-way ANOVA evaluated for effects
of spaceflight (i.e., Ground vs. flight) and treatment (i.e., IgG vs. YN41), where a Dunnett’s
post-hoc test compared each treatment group to IgG Ground Control (where groups not con-
nected by the same letter are significantly different); α= 0.05.
Microarchitectural assessment of trabecular and cortical bone at the distal femur demon-
strated significant bone deterioration with microgravity exposure with no effect of myostatin
inhibition (Table 2;Fig 6). IgG ground control mice demonstrated skeletal growth, via
increased bone geometric measures, as compared to their younger, Baseline counterparts pri-
marily in cortical, and not trabecular, bone with significant increases in BA, Ct.Th, maximum
and minimum cross-sectional moments of inertia, and volumetric bone mineral density in
cortical bone, along with significantly decreased cortical porosity. In addition, both cortical
and trabecular bone were affected by unloading in microgravity with no significant effect of
myostatin inhibition. Comparisons showed a consistent pattern of diminished cortical and tra-
becular bone microarchitecture across all standard μCT measures following microgravity
exposure (Table 2).
Finally, three-point flexural testing was used to assess the influence of microgravity-induced
bone decrements on mechanical properties (Table 3). Despite the decrease in BMD seen in the
femurs of flight mice, stiffness, energy to break, peak load and ultimate elongation parameters
were not significantly affected compared to their ground counterparts. Myostatin inhibition
had no effect on biomechanical properties (Table 3).
Discussion
Rodent Research-3, sponsored by the ISS National Laboratory, launched to the ISS aboard
NASA’s eighth cargo resupply flight of the SpaceX Dragon spacecraft (SpX-8) on April 8, 2016.
The primary goal of the RR-3 investigation was to determine if inhibition of myostatin,
through delivery of a neutralizing anti-myostatin antibody, could prevent the loss of skeletal
muscle mass that was expected to occur in mice under conditions of microgravity. The live
phase of the experiment was one of the longest to date, being 6 weeks in length, and involved
female BALB/C mice that were 12 weeks at the time of launch. Both interim and terminal mea-
sures were collected, made possible by new onboard anesthesia recovery hardware and proce-
dures. The mission was also unique in that it measured muscle function with a grip strength
meter during flight, and so for the first time could evaluate longitudinal functional conse-
quences of microgravity disuse in addition to endpoint changes in muscle mass.
Microgravity conditions resulted in overall loss of muscle mass and underlying bone in the
flight mice compared to ground controls. Total carcass weight was also decreased with space-
flight, although not significantly. Lean mass loss was apparent by 4 weeks as evidenced by
interim DEXA measures and progressed through 6 weeks of microgravity exposure. Of the 7
individual muscles collected at termination, the soleus muscle atrophied the most, losing
28.4% of weight compared to the ground controls and falling below baseline levels. Others
have reported a similar dramatic atrophy of this type I slow-twitch postural muscle during
spaceflight as measured by reduced myofiber CSA [8,11,13]. The predominantly fast-twitch
type II gastrocnemius muscle also showed significant loss (-12.5%) in mass compared to
ground controls consistent with previous histological findings [8]. The EDL showed no evi-
dence of wasting, consistent with the reported maintenance of gene expression [11] and myofi-
ber CSA [13] following spaceflight. Similarly, EDL mass was unchanged in hindlimb
suspended mice in 1g [38]. In contrast, grip strength measurements demonstrated that muscle
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Fig 6. Representative micro-computed tomography images from the distal femur from baselines as well as ground and flight mice treated with
either IgG or YN41. While visual differences between groups are subtle, the microarchitecture of both trabecular and cortical compartments
significantly improved with skeletal maturation when comparing baseline to ground controls (IgG), and both bone compartments were significantly
diminished with microgravity exposure (comparing IgG ground to flight groups) but were unaffected by myostatin inhibition.
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strength in flight animals was decreased by 15.6% more than ground controls at 4 weeks into
flight. This is the first direct evidence of muscle weakness in mice as measured during space-
flight. Microgravity also affected underlying bone. In vivo DEXA showed a decrease in hin-
dlimb BMD and ex vivo measures confirmed a decrease in distal and mid-femur BMD.
Microarchitectural assessment of cortical and trabecular bone at the distal femur showed con-
sistent detrimental influences of microgravity exposure, yet no influence of the myostatin inhi-
bition. Despite these decreases, no significant effects on tibial biomechanical properties were
seen in flight animals compared to their ground counterparts. Lumbar vertebral areal BMD
was not affected by spaceflight. Mice were not skeletally mature throughout the duration of
this study [3941], where skeletal maturity is generally considered to occur around 20 weeks of
age. Therefore it is possible that ongoing bone formation was sufficient to mitigate the cata-
bolic effects of microgravity exposure in these growing, female BALB/c mice over the 6-week
duration of the live phase study. However, other studies in similarly aged female BALB/c and
C57BL/6 mice reveal significant losses after two-weeks of microgravity exposure [12,18].
These results suggest the need for future studies to evaluate the effects of age, duration of
microgravity exposure, and how genetic background may differently affect bone in spaceflight.
Inhibiting the growth factor myostatin with a neutralizing antibody has been shown in a
recent proof-of-concept clinical trial to be able to increase lean mass and power measures in
an elderly weak population [27]. The humanized myostatin antibody used in that trial was
derived from the murine antibody used in this study (YN41, LSN2478185). As shown in earlier
work with rodent models [26], myostatin inhibition was able to increase body weight, lean
mass, individual muscle weights and muscle strength in mice under regular ground condi-
tions. The effect of myostatin inhibition in this study was significant on predominantly fast-
twitch muscles (ranging from an increase of 31.6% in weight for the triceps to 12.9% for the
TA), with a trend for an increase of 3.6% increase for the slow-twitch type I soleus muscle.
This is consistent with previous reports of myostatin inhibition [26]. Notably, myostatin and
ActRIIb mRNA levels are greater in fast- than in slow-twitch muscles [42]. In this study, myos-
tatin inhibition was able to increase lean mass, muscle weights and muscle strength in flight
animals compared to the IgG flight controls in an overall manner similar to the effect seen on
ground control animals, demonstrating that muscle building effects of myostatin inhibition
including increases in function do not require the weight-bearing effects of gravity. In fact, the
myostatin antibody was able to prevent all of the losses in hindlimb lean mass, grip strength
and muscle weights (with the exception of the soleus) induced by microgravity. For some indi-
vidual muscles e.g., quadriceps, triceps, TA, plantaris, myostatin inhibition significantly
increased weight over that of ground IgG controls, demonstrating that YN41 was not only able
to overcome the muscle atrophy induced by spaceflight but induce even larger effects. Mice in
the NASA flight hardware modules exhibit species-typical behaviors including running (i.e.,
Table 3. Effect of microgravity and myostatin inhibition on femoral biomechanics.
Femoral Biomechanics
Group Stiffness (kgf/mm) Peak Load (kgf) Energy to Break (kgf x mm/mm
3
) Ultimate Elongation (mm)
Baseline 8.7±0.45 1.7±0.07 0.28±0.01 0.27±0.01
Ground IgG 9.9±0.45 1.9±0.06 0.30±0.02 0.27±0.01
Ground YN41 9.9±0.25 1.9±0.04 0.35±0.02 0.29±0.01
Flight IgG 9.4±0.18 1.8±0.04 0.30±0.02 0.27±0.01
Flight YN41 9.7±0.43 1.9±0.05 0.29±0.01 0.26±0.01
Data are shown ±s.e.m.
https://doi.org/10.1371/journal.pone.0230818.t003
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Myostatin inhibition prevents microgravity-induced loss of skeletal muscle mass and strength
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circularly about the cage) and grasping the wire mesh walls, the ground control mice are also
active [43]. The triceps brachii muscles may provide insight into relative activity differences
between flight and ground mice; however, these masses do not explain the increased grip
strength of mice treated with the antibody (e.g., regression of grip strength change by 6 weeks
poorly correlates with triceps brachii masses, R
2
= 0.15). However, myostatin antibody resulted
in myofiber hypertrophy, as measured by myofiber CSA, as expected. As on the ground, effects
of myostatin inhibition were larger on fast-twitch muscles than on the slow-twitch soleus
muscle.
However, some differences in response to myostatin inhibition between flight and ground
animals were noticeable. The increase in hind limb lean mass and muscle strength induced by
myostatin inhibition was not as large in flight animals as compared to ground controls, sup-
ported by the plantaris (24.9% ground vs 18.7% flight). However, in contrast, the weights of
the TA and soleus muscles were increased more in YN41-treated flight animals compared to
YN41-treated ground controls (12.9% ground vs 26.9% flight for TA, 3.6% ground vs 11.2%
flight, versus their respective IgG controls). The reason for these differences in responsiveness
is unclear and needs to be confirmed.
At the molecular level microgravity was shown to result in decreases in RNA encoding
muscle cytoskeletal proteins Actc1 and Mybph. This is consistent with the reduction in muscle
mass that had occurred in the skeletal muscles. Trim63 (MurF1) expression increased in space
consistent with the activation of the ubiquitin-proteosome pathway to induce muscle atrophy
[44]. Changes in Frzd9 and Kcnma1 gene expression induced by spaceflight appear robust
since these changes reproduced those previously reported [11,36]. Lack of reproducibility of
changes in expression of other genes reported by others could be due to differences in space-
flight duration or mouse strain. Not surprisingly, many of the directional changes in gene
expression induced by spaceflight seen in type II muscles (i.e., gastrocnemius, quadriceps, TA)
were not seen in the type I soleus muscle (e.g., Actc1) and vice-a-versa (e.g., Acrv2b and Alk4).
Regarding the effects of myostatin inhibition on gene expression, the changes observed in the
28 genes examined were more plentiful than microgravity, perhaps not surprising since the
effect of YN41 treatment on muscle weights was larger–at least for fast twitch muscles. Myosta-
tin inhibition does ‘reverse’ some of the effects of microgravity on underlying muscle gene
expression, notably Actc1 and Mybph. Spaceflight reduces expression of these genes and
YN41 treatment increased their expression. As these are genes that encode proteins involved
in muscle architecture, it might be expected that microgravity should reduce expression and
myostatin inhibition should increase. However, more often it appears that myostatin inhibi-
tion modulates genes that are not affected by microgravity and vice-a-versa. For example,
Itgb5, Fst and Cyr61gene expression was consistently reduced by YN41 but these genes were
not affected by spaceflight. It is of interest to note that Cyr61 is increased in human skeletal
muscle after mechanical loading [45]. The decrease in Fst induced by myostatin inhibition is
perhaps an attempt to compensate for the decreased levels of active myostatin by reducing lev-
els of one of its natural inhibitors. The increase in expression of Gamt both in YN41-treated
flight and ground mice is interesting and may contribute to the increase grip strength mea-
sured in these groups as Gamt deficient mice have been shown to have lower force than their
normal counterparts [46,47]. The decrease in Retn [46] with YN41 treatment in both flight
and ground groups is novel and the decrease in resistin levels with increased muscle strength
due to resistance training in the elderly is of note.
Myostatin inhibition was not able to reverse bone loss that was observed in flight animals,
despite the dramatic increase induced in muscle mass with treatment. Here, spaceflight
resulted in diminished bone mass and microarchitecture, yet myostatin inhibition did not
show any significant effect on BMD even with the weight-bearing effect of increased muscle
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Myostatin inhibition prevents microgravity-induced loss of skeletal muscle mass and strength
PLOS ONE | https://doi.org/10.1371/journal.pone.0230818 April 21, 2020 19 / 23
mass in ground controls. This was not unexpected as myostatin inhibition did not show any
significant effect on BMD even with the weight-bearing effect of increased muscle mass in
ground controls and was consistent with previous observations [48]. While it is possible that
increased muscle mass and strength may have had an effect on bone outcome measures, and
in particular microCT assessment of the distal long bones, we observed no such relationships
in the present study or in a prior study of female C57BL/6 mice that were flown on the Space
Shuttle for ~two weeks (unpublished data). It is likely that the increased loading from muscles
of greater mass and strength following myostatin inhibition caused small, but insignificant
increases in bone mass. Site-specific assessment of bone microarchitecture and material prop-
erties (e.g., via nanomechanical testing) would be needed to evaluate for bone changes proxi-
mal to sites of muscle attachments. Also, a longer duration experiment could have allowed the
more slowly adapting bone tissue adjust to increased skeletal muscle mass and strength.
The mice in flight, regardless of treatment group, adapted well to their new environment
and appeared healthy through the 6 weeks of the experiment. However, the flight mice did
exhibit ‘racetrack’ behavior by 2 weeks [43], which would likely provide physical activity and
some degree of musculoskeletal loading during spaceflight. However, there was no detectable
effect of this running behavior or gripping the wire mesh walls of the NASA habitats on the tri-
ceps brachii masses or grip strength. It is noteworthy that heart weight was reduced in the
Flight IgG group compared to ground controls. Myostatin inhibition was able to prevent that
loss in flight animals, although on the ground the myostatin antibody-treated group had no
effect on heart weight. Myostatin inhibition has previously been shown to not affect heart
weight.
In summary, mice in space are a useful model of muscle atrophy since effects are global and
can be maintained for many weeks if not months, unlike many ground models of muscle wast-
ing. Myostatin inhibition prevented the muscle atrophy and accompanying muscle weakness
seen in mice that were flown aboard the ISS for six weeks, one of the longest rodent flights to
date, thus demonstrating myostatin inhibition as an effective countermeasure to this detri-
mental consequence of life under microgravity conditions. A number of myostatin pathway
inhibitors are in clinical trials for muscle atrophy and have been shown to increase muscle
mass and power measures in man [27,4951]. This raises the future possibility in astronauts
and cosmonauts of therapeutic intervention to counteract muscle atrophy induced by long
term spaceflight and may be important for successful missions to the Moon and Mars.
Supporting information
S1 Table.
(DOCX)
S2 Table. Gene expression values for gastronemius, quadriceps, soleus and tibialis anterior
muscles from flight and ground groups.
(DOCX)
S1 Raw Data.
(XLSX)
Acknowledgments
Support of the Techshot experts, Richard Boling, John Vellinger and Dr. Paul Todd, was
deeply appreciated. Dennis Leveson-Gower, the Project Scientist, along with the NASA Rodent
Research team, at NASA Ames Research Center were instrumental in the successful execution
of the study. NASA Kennedy Space Center animal care and laboratory support were truly
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Myostatin inhibition prevents microgravity-induced loss of skeletal muscle mass and strength
PLOS ONE | https://doi.org/10.1371/journal.pone.0230818 April 21, 2020 20 / 23
outstanding. We greatly appreciate the excellent support of the flight crew, Timothy Peake and
Timothy Kopra. Finally, we acknowledge the excellent direct crew flight operations support
from Shankini Doraisingam and other support from BioServe Space Technologies at the Uni-
versity of Colorado Boulder.
Author Contributions
Conceptualization: Rosamund C. Smith, Martin S. Cramer, Louis S. Stodieck.
Data curation: Rosamund C. Smith, Martin S. Cramer, Pamela J. Mitchell, Jonathan Lucchesi,
Alicia M. Ortega, Eric W. Livingston, Darryl Ballard, Ling Zhang, Jeff Hanson, Kenneth
Barton, Shawn Berens, Kelly M. Credille, Ted A. Bateman, Virginia L. Ferguson, Yanfei L.
Ma, Louis S. Stodieck.
Formal analysis: Rosamund C. Smith, Martin S. Cramer, Pamela J. Mitchell, Jonathan Luc-
chesi, Eric W. Livingston, Darryl Ballard, Ling Zhang, Jeff Hanson, Kenneth Barton, Shawn
Berens, Kelly M. Credille, Ted A. Bateman, Virginia L. Ferguson, Yanfei L. Ma, Louis S.
Stodieck.
Investigation: Rosamund C. Smith, Martin S. Cramer, Pamela J. Mitchell, Jonathan Lucchesi,
Alicia M. Ortega, Eric W. Livingston, Darryl Ballard, Ling Zhang, Jeff Hanson, Shawn
Berens, Kelly M. Credille, Ted A. Bateman, Virginia L. Ferguson, Yanfei L. Ma, Louis S.
Stodieck.
Methodology: Pamela J. Mitchell, Alicia M. Ortega.
Supervision: Rosamund C. Smith, Yanfei L. Ma, Louis S. Stodieck.
Writing – original draft: Rosamund C. Smith.
Writing – review & editing: Martin S. Cramer, Pamela J. Mitchell, Jonathan Lucchesi, Alicia
M. Ortega, Eric W. Livingston, Darryl Ballard, Ling Zhang, Jeff Hanson, Kenneth Barton,
Shawn Berens, Kelly M. Credille, Ted A. Bateman, Virginia L. Ferguson, Yanfei L. Ma,
Louis S. Stodieck.
References
1. Lang T. et al. Cortical and trabecular bone mineral loss from the spine and hip in long-duration space-
flight. J. Bone Miner. Res. 19, 1006–1012 (2004). https://doi.org/10.1359/JBMR.040307 PMID:
15125798
2. LeBlanc A. et al. Bone mineral and lean tissue loss after long duration space flight. J Musculoskelet
Neuronal Interact 1, 157–160 (2000). PMID: 15758512
3. Chang D. G. et al. Lumbar Spine Paraspinal Muscle and Intervertebral Disc Height Changes in Astro-
nauts After Long-Duration Spaceflight on the International Space Station. Spine 41, 1917–1924 (2016).
https://doi.org/10.1097/BRS.0000000000001873 PMID: 27779600
4. Fitts R. H. et al. Prolonged space flight-induced alterations in the structure and function of human skele-
tal muscle fibres. J. Physiol. (Lond.) 588, 3567–3592 (2010).
5. Tesch P. A., et al. Effects of 17-day spaceflight on knee extensor muscle function and size. Eur. J. Appl.
Physiol. 93, 463–468 (2005). https://doi.org/10.1007/s00421-004-1236-9 PMID: 15517339
6. Vico L. et al. Cortical and Trabecular Bone Microstructure Did Not Recover at Weight-Bearing Skeletal
Sites and Progressively Deteriorated at Non-Weight-Bearing Sites During the Year Following Interna-
tional Space Station Missions. J. Bone Miner. Res. 32, 2010–2021 (2017). https://doi.org/10.1002/
jbmr.3188 PMID: 28574653
7. Lalani R. et al. Myostatin and insulin-like growth factor-I and -II expression in the muscle of rats exposed
to the microgravity environment of the NeuroLab space shuttle flight. J. Endocrinol. 167, 417–428
(2000). https://doi.org/10.1677/joe.0.1670417 PMID: 11115768
PLOS ONE
Myostatin inhibition prevents microgravity-induced loss of skeletal muscle mass and strength
PLOS ONE | https://doi.org/10.1371/journal.pone.0230818 April 21, 2020 21 / 23
8. Harrison B. C. et al. Skeletal muscle adaptations to microgravity exposure in the mouse. Journal of
Applied Physiology 95, 2462–2470 (2003). https://doi.org/10.1152/japplphysiol.00603.2003 PMID:
12882990
9. Sung M. et al. Spaceflight and hind limb unloading induce similar changes in electrical impedance char-
acteristics of mouse gastrocnemius muscle. Journal of Musculoskeletal Neuronal Interactions 13,
(2013).
10. Shen H. et al. Effects of spaceflight on the muscles of the murine shoulder. FASEB J. 31, 5466–5477
(2017). https://doi.org/10.1096/fj.201700320R PMID: 28821629
11. Gambara G. et al. Gene Expression Profiling in Slow-Type Calf Soleus Muscle of 30 Days Space-Flown
Mice. PLoS ONE 12, e0169314 (2017). https://doi.org/10.1371/journal.pone.0169314 PMID:
28076365
12. Radugina E. A. et al. Exposure to microgravity for 30 days onboard Bion M1 caused muscle atrophy
and impaired regeneration in murine femoral Quadriceps. Life Sci Space Res (Amst) 16, 18–25 (2018).
13. SandonàD. et al. Adaptation of mouse skeletal muscle to long-term microgravity in the MDS mission.
PLoS ONE 7, e33232 (2012). https://doi.org/10.1371/journal.pone.0033232 PMID: 22470446
14. Philippou A. et al. Masticatory muscles of mouse do not undergo atrophy in space. FASEB J. 29, 2769–
2779 (2015). https://doi.org/10.1096/fj.14-267336 PMID: 25795455
15. Tavella S. et al. Bone turnover in wild type and pleiotrophin-transgenic mice housed for three months in
the International Space Station (ISS). PLoS ONE 7, e33179 (2012). https://doi.org/10.1371/journal.
pone.0033179 PMID: 22438896
16. Berg-Johansen B. et al. Spaceflight-induced bone loss alters failure mode and reduces bending
strength in murine spinal segments. J. Orthop. Res. 34, 48–57 (2016). https://doi.org/10.1002/jor.
23029 PMID: 26285046
17. Shiba D. et al. Development of new experimental platform ‘MARS’-Multiple Artificial-gravity Research
System-to elucidate the impacts of micro/partial gravity on mice. Sci Rep 7, 10837 (2017). https://doi.
org/10.1038/s41598-017-10998-4 PMID: 28883615
18. Lloyd S. A. et al. Osteoprotegerin is an effective countermeasure for spaceflight-induced bone loss in
mice. Bone 81, (2015).
19. Gerbaix M. et al. One-month spaceflight compromises the bone microstructure, tissue-level mechanical
properties, osteocyte survival and lacunae volume in mature mice skeletons. Sci Rep 7, 2659 (2017).
https://doi.org/10.1038/s41598-017-03014-2 PMID: 28572612
20. Kalyani R. R., et al. Age-related and disease-related muscle loss: the effect of diabetes, obesity, and
other diseases. Lancet Diabetes Endocrinol 2, 819–829 (2014). https://doi.org/10.1016/S2213-8587
(14)70034-8 PMID: 24731660
21. Legrand D. et al. Muscle strength and physical performance as predictors of mortality, hospitalization,
and disability in the oldest old. J Am Geriatr Soc 62, 1030–1038 (2014). https://doi.org/10.1111/jgs.
12840 PMID: 24802886
22. Bharucha-Goebel D. & Kaufmann P. Treatment advances in spinal muscle atrophy. Curr Neurol Neu-
rosci Rep. 17(11):91 (2017). https://doi.org/10.1007/s11910-017-0798-y PMID: 28983837
23. Trappe S. et al. Exercise in space: human skeletal muscle after 6 months aboard the International
Space Station. J. Appl. Physiol. 106, 1159–1168 (2009). https://doi.org/10.1152/japplphysiol.91578.
2008 PMID: 19150852
24. Smith R. C. & Lin B. K. Myostatin inhibitors as therapies for muscle wasting associated with cancer and
other disorders. Curr Opin Support Palliat Care 7, 352–360 (2013). https://doi.org/10.1097/SPC.
0000000000000013 PMID: 24157714
25. Elkasrawy M. N. & Hamrick M. W. Myostatin (GDF-8) as a key factor linking muscle mass and bone
structure. J Musculoskelet Neuronal Interact 10, 56–63 (2010). PMID: 20190380
26. Smith R. C. et al. Myostatin Neutralization Results in Preservation of Muscle Mass and Strength in Pre-
clinical Models of Tumor-Induced Muscle Wasting. Mol. Cancer Ther. 14, 1661–1670 (2015). https://
doi.org/10.1158/1535-7163.MCT-14-0681 PMID: 25908685
27. Becker C. et al. Myostatin antibody (LY2495655) in older weak fallers: a proof-of-concept, randomised,
phase 2 trial. Lancet Diabetes Endocrinol 3, 948–957 (2015). https://doi.org/10.1016/S2213-8587(15)
00298-3 PMID: 26516121
28. Sun G.-S., et al. The past, present, and future of National Aeronautics and Space Administration space-
flight diet in support of microgravity rodent experiments. Nutrition 30, 125–130 (2014). https://doi.org/
10.1016/j.nut.2013.04.005 PMID: 24012282
29. Moyer E. L. et al. Evaluation of rodent spaceflight in the NASA animal enclosure module for an extended
operational period (up to 35 days). NPJ Microgravity 2, 16002 (2016). https://doi.org/10.1038/
npjmgrav.2016.2 PMID: 28725722
PLOS ONE
Myostatin inhibition prevents microgravity-induced loss of skeletal muscle mass and strength
PLOS ONE | https://doi.org/10.1371/journal.pone.0230818 April 21, 2020 22 / 23
30. Deacon R. M. Measuring the strength of mice. J Vis Exp. 2013 Jun 2;(76).
31. Smith R.C. et al. Circulating αKlotho influences phosphate handling by controlling FGF23 production. J
Clin Invest. 122(12):4710–5 (2012). https://doi.org/10.1172/JCI64986 PMID: 23187128
32. Sabsovich I. et al. Bone microstructure and its associated genetic variability in 12 inbred mouse strains:
microCT study and in silico genome scan. Bone. 42(2):439–51 (2008). https://doi.org/10.1016/j.bone.
2007.09.041 PMID: 17967568
33. Bouxsein M. L. et al. Guidelines for assessment of bone microstructure in rodents using micro-com-
puted tomography. J. Bone Miner. Res. 25, 1468–1486 (2010). https://doi.org/10.1002/jbmr.141 PMID:
20533309
34. Okragly A. J. et al. Elevated levels of Interleukin (IL)-33 induce bone pathology but absence of IL-33
does not negatively impact normal bone homeostasis. Cytokine 79, 66–73 (2016). https://doi.org/10.
1016/j.cyto.2015.12.011 PMID: 26771472
35. Fitzgerald J. Cartilage breakdown in microgravity-a problem for long-term spaceflight? NPJ Regen Med
2, 10 (2017). https://doi.org/10.1038/s41536-017-0016-1 PMID: 29302346
36. Allen D. L. et al. Myonuclear number and myosin heavy chain expression in rat soleus single muscle
fibers after spaceflight. J. Appl. Physiol. 81, 145–151 (1996). https://doi.org/10.1152/jappl.1996.81.1.
145 PMID: 8828656
37. Latres E. et al. Myostatin blockade with a fully human monoclonal antibody induces muscle hypertrophy
and reverses muscle atrophy in young and aged mice. Skelet Muscle 5, 34 (2015). https://doi.org/10.
1186/s13395-015-0060-8 PMID: 26457176
38. Hanson A. M., Harrison B. C., Young M. H., Stodieck L. S. & Ferguson V. L. Longitudinal characteriza-
tion of functional, morphologic, and biochemical adaptations in mouse skeletal muscle with hindlimb
suspension. Muscle and Nerve 48, (2013).
39. Ferguson V. L., Ayers R. A., Bateman T. A. & Simske S. J. Bone development and age-related bone
loss in male C57BL/6J mice. Bone 33, (2003).
40. Glatt V., Canalis E., Stadmeyer L. & Bouxsein M. L. Age-related changes in trabecular architecture dif-
fer in female and male C57BL/6J mice. in Journal of Bone and Mineral Research 22, 1197–1207 ( John
Wiley & Sons, Ltd, 2007). https://doi.org/10.1359/jbmr.070507 PMID: 17488199
41. Willinghamm M. D. et al. Age-Related Changes in Bone Structure and Strength in Female and Male
BALB/c Mice. Calcif Tissue Int 86, 470–483 (2010). https://doi.org/10.1007/s00223-010-9359-y PMID:
20405109
42. Allen D. L. & Unterman T. G. Regulation of myostatin expression and myoblast differentiation by FoxO
and SMAD transcription factors. American journal of physiology. Cell physiology 292, NaN-NaN
(2006).
43. Ronca A. E. et al. Behavior of mice aboard the International Space Station. Scientific Reports 9, 4717
(2019). https://doi.org/10.1038/s41598-019-40789-y PMID: 30976012
44. R Bodine S. C. & Baehr L. M. Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/
atrogin-1. Am J Physiol Endocrinol Metab. 307(6):E469–84 (2014). https://doi.org/10.1152/ajpendo.
00204.2014 PMID: 25096180
45. Kivela
¨R. et al. A single bout of exercise with high mechanical loading induces the expression of Cyr61/
CCN1 and CTGF/CCN2 in human skeletal muscle. J. Appl. Physiol. 103, 1395–1401 (2007). https://
doi.org/10.1152/japplphysiol.00531.2007 PMID: 17673559
46. Prestes J. et al. The Effects of Muscle Strength Responsiveness to Periodized Resistance Training on
Resistin, Leptin, and Cytokine in Elderly Postmenopausal Women. J Strength Cond Res 32, 113–120
(2018). https://doi.org/10.1519/JSC.0000000000001718 PMID: 28661971
47. Kan H. E. et al. Lower force and impaired performance during high-intensity electrical stimulation in
skeletal muscle of GAMT-deficient knockout mice. Am. J. Physiol., Cell Physiol. 289, C113–119
(2005). https://doi.org/10.1152/ajpcell.00040.2005 PMID: 15743892
48. Arounleut P. et al. A myostatin inhibitor (propeptide-Fc) increases muscle mass and muscle fiber size in
aged mice but does not increase bone density or bone strength. Experimental Gerontology 48, 898–
904 (2013). https://doi.org/10.1016/j.exger.2013.06.004 PMID: 23832079
49. Rooks D. S. et al. Effect of bimagrumab on thigh muscle volume and composition in men with casting-
induced atrophy. J Cachexia Sarcopenia Muscle 8, 727–734 (2017). https://doi.org/10.1002/jcsm.
12205 PMID: 28905498
50. Garito T. et al. Bimagrumab improves body composition and insulin sensitivity in insulin-resistant individu-
als. Diabetes Obes Metab 20, 94–102 (2018). https://doi.org/10.1111/dom.13042 PMID: 28643356
51. Rooks D. et al. Treatment of Sarcopenia with Bimagrumab: Results from a Phase II, Randomized, Con-
trolled, Proof-of-Concept Study. J Am Geriatr Soc 65, 1988–1995 (2017). https://doi.org/10.1111/jgs.
14927 PMID: 28653345
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... 1−5 A fast deterioration of skeletal muscle tissue, in particular, affects astronauts who are exposed both to gravitational unloading (hereafter termed microgravity or "μg") and to cosmic radiation during spaceflight. For this reason, a variety of strategies for muscular maintenance in vitro and in vivo has also been devised for terrestrial benefit, including physical exercise, 6 mechanical stimulation in the form of vibrations 7 and pressure application, 1 electrical stimulation, 8 exposure to hypergravity, 9−11 administration of soluble factors (such as activin type IIB receptor, 12 recombinant myokine irisin, 13 and myostatin antibody YN41 14 ), and even genetic transduction finalized to the overexpression of nucleic acids (such as a long noncoding RNA termed lncMUMA). 15 A common approach against skeletal muscle waste due to mechanical unloading also consists of the supply of antioxidant compounds, like for instance (-)-epicatechin, 16 lecithin, 17 N-acetylcysteine, 18 complex mixtures of polyphenols associated with other antioxidants (such as vitamin E, selenium, and omega-3 fatty acids), 19 or even seed extracts (from Oenothera odorata). ...
... 15 A common approach against skeletal muscle waste due to mechanical unloading also consists of the supply of antioxidant compounds, like for instance (-)-epicatechin, 16 lecithin, 17 N-acetylcysteine, 18 complex mixtures of polyphenols associated with other antioxidants (such as vitamin E, selenium, and omega-3 fatty acids), 19 or even seed extracts (from Oenothera odorata). 20 Besides targeting autophagic flux 21 and myostatin signaling, 2,10,12,14 the most recent research indeed focuses on the role of oxidative stress (OS) due to excess reactive oxygen species (ROS) and mitochondrial dysregulation in skeletal muscle degeneration under real or simulated microgravity (sμg). 12,18,22,23 As human permanence in the low Earth orbit undergoes increasing duration and opens to interplanetary travel, the understanding of the biological effects of mechanical unloading necessitates deepening by careful consideration of the consequences of exposure to highly energetic cosmic radiations that to date is largely obscure and apparently follows divergent molecular pathways in comparison to microgravity. ...
... Some studies have identified altered levels of proteins critical to muscle regeneration in astronauts, such as myostatin, activin A, and certain cytokines (e.g. IL-6, IL-10, IL-1ra), which may represent potential targets for pharmacological interventions (Gertz et al., 2020;Lee et al., 2020b;Smith et al., 2020). At present, resistance exercise is the main countermeasure for preventing muscle atrophy and bone loss. ...
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Recent advancements in next generation spacecrafts have reignited public excitement over life beyond Earth. However, to safeguard the health and safety of humans in the hostile environment of space, innovation in pharmaceutical manufacturing and drug delivery deserves urgent attention. In this review/commentary, the current state of medicines provision in space is explored, accompanied by a forward look on the future of pharmaceutical manufacturing in outer space. The hazards associated with spaceflight, and their corresponding medical problems, are first briefly discussed. Subsequently, the infeasibility of present-day medicines provision systems for supporting deep space exploration is examined. The existing knowledge gaps on the altered clinical effects of medicines in space are evaluated, and suggestions are provided on how clinical trials in space might be conducted. An envisioned model of on-site production and delivery of medicines in space is proposed, referencing emerging technologies (e.g. Chemputing, synthetic biology, and 3D printing) being developed on Earth that may be adapted for extra-terrestrial use. This review concludes with a critical analysis on the regulatory considerations necessary to facilitate the adoption of these technologies and proposes a framework by which these may be enforced. In doing so, this commentary aims to instigate discussions on the pharmaceutical needs of deep space exploration, and strategies on how these may be met.
... The myostatin signaling pathway is a key negative regulator of muscle mass which affects SC proliferation and differentiation 43 and has been targeted to protect against skeletal muscle atrophy during spaceflight 106 . In rodent models, inhibition of myostatin using a neutralizing antibody has a protective effect against loss of skeletal muscle mass and strength during spaceflight 106,164 . Myostatin is downregulated endogenously with exercise; for example, Cotter et al. 34 showed that concurrent high intensity interval aerobic exercise and maximal exertion strength training could mitigate the increase in myostatin during simulated µG with limb suspension. ...
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During spaceflight missions, astronauts work in an extreme environment with several hazards to physical health and performance. Exposure to microgravity results in remarkable deconditioning of several physiological systems, leading to impaired physical condition and human performance, posing a major risk to overall mission success and crew safety. Physical exercise is the cornerstone of strategies to mitigate physical deconditioning during spaceflight. Decades of research have enabled development of more optimal exercise strategies and equipment onboard the International Space Station. However, the effects of microgravity cannot be completely ameliorated with current exercise countermeasures. Moreover, future spaceflight missions deeper into space require a new generation of spacecraft, which will place yet more constraints on the use of exercise by limiting the amount, size and weight of exercise equipment and the time available for exercise. Space agencies are exploring ways to optimise exercise countermeasures for spaceflight, specifically exercise strategies that are more efficient, require less equipment and are less time-consuming. Blood flow restriction exercise is a low intensity exercise strategy that requires minimal equipment and can elicit positive training benefits across multiple physiological systems. This method of exercise training has potential as a strategy to optimise exercise countermeasures during spaceflight and reconditioning in terrestrial and partial gravity environments. The possible applications of blood flow restriction exercise during spaceflight are discussed herein.
... The myostatin signaling pathway is a key negative regulator of muscle mass which affects SC proliferation and differentiation 43 and has been targeted to protect against skeletal muscle atrophy during spaceflight 106 . In rodent models, inhibition of myostatin using a neutralizing antibody has a protective effect against loss of skeletal muscle mass and strength during spaceflight 106,164 . Myostatin is downregulated endogenously with exercise; for example, Cotter et al. 34 showed that concurrent high intensity interval aerobic exercise and maximal exertion strength training could mitigate the increase in myostatin during simulated µG with limb suspension. ...
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INTRODUCTION: During spaceflight missions, astronauts work in an extreme environment with several hazards to physical health and performance. Exposure to microgravity results in remarkable deconditioning of several physiological systems, leading to impaired physical condition and human performance, posing a major risk to overall mission success and crew safety. Physical exercise is the cornerstone of strategies to mitigate physical deconditioning during spaceflight. Decades of research have enabled development of more optimal exercise strategies and equipment onboard the International Space Station. However, the effects of microgravity cannot be completely ameliorated with current exercise countermeasures. Moreover, future spaceflight missions deeper into space require a new generation of spacecraft, which will place yet more constraints on the use of exercise by limiting the amount, size, and weight of exercise equipment and the time available for exercise. Space agencies are exploring ways to optimize exercise countermeasures for spaceflight, specifically exercise strategies that are more efficient, require less equipment, and are less time-consuming. Blood flow restriction exercise is a low intensity exercise strategy that requires minimal equipment and can elicit positive training benefits across multiple physiological systems. This method of exercise training has potential as a strategy to optimize exercise countermeasures during spaceflight and reconditioning in terrestrial and partial gravity environments. The possible applications of blood flow restriction exercise during spaceflight are discussed herein.Hughes L, Hackney KJ, Patterson SD. Optimization of exercise countermeasures to spaceflight using blood flow restriction. Aerosp Med Hum Perform. 2021; 93(1):32-45.
... Smith et al. measured in a 90 h ISS experiment with a group of mice, a lower expression in the RNA encoding muscle cytoskeletal proteins Actc1 and Mybph [75]. Guanidinoacetate methyltransferase (Gamt), an N-methyltransferase that catalyzes synthesis of creatine, was significantly increased by spaceflight in the quadriceps, whereas Myosin binding protein H (Mybph) was significantly decreased by µg in the gastrocnemius. ...
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Introduction : A long-term stay of humans in space causes a large number of well-known health problems and changes in protists and plants. Deep space exploration will increase the time humans or rodents will spend in microgravity (µg). Moreover, they are exposed to cosmic radiation, hypodynamia, and isolation. OMICS investigations will increase our knowledge of the underlying mechanisms of µg-induced alterations in vivo and in vitro. Areas covered : We summarize the findings over the recent 3 years on µg-induced changes in the proteome of protists, plants, rodent and human cells. Considering the thematic orientation of microgravity-related publications in that time frame, we focus on medicine-associated findings such as the µg-induced antibiotic resistance of bacteria, the myocardial consequences of µg-induced calpain activation and the role of MMP13 in osteoarthritis. All these point to the fact that µg is an extreme stressor that could not be evolutionarily addressed on Earth. Expert Commentary : In conclusion, when interpreting µg-experiments, the direct, mostly unspecific stress response, must be distinguished from specific µg-effects. For this reason, recent studies often do not consider single protein findings but place them in the context of protein-protein interactions. This enables an estimation of functional relationships, especially if these are supported by epigenetic and transcriptional data (multi-omics).
... Thus, microgravity-induced muscle loss provides an opportunity to study muscle-wasting progression on a faster timescale than is possible on Earth. Multiple studies have been conducted using rodents in microgravity as an accelerated disease model to elucidate mechanisms underlying muscle atrophy and to test new potential therapeutics (Chakraborty et al., 2020;da Silveira et al., 2020;Lawler et al., 2021;Semple et al., 2020;Smith et al., 2020). Moving beyond rodent models, microgravity provides a unique opportunity to study sarcopenia and disuse atrophy in human cellular models. ...
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Research in low Earth orbit (LEO) has become more accessible. The 2020 Biomanufacturing in Space Symposium reviewed space-based regenerative medicine research and discussed leveraging LEO to advance biomanufacturing for regenerative medicine applications. The symposium identified areas where financial investments could stimulate advancements overcoming technical barriers. Opportunities in disease modeling, stem-cell-derived products, and biofabrication were highlighted. The symposium will initiate a roadmap to a sustainable market for regenerative medicine biomanufacturing in space. This perspective summarizes the 2020 Biomanufacturing in Space Symposium, highlights key biomanufacturing opportunities in LEO, and lays the framework for a roadmap to regenerative medicine biomanufacturing in space.
... Additionally, alterations in gene expression related to monoamine turnover and D1-receptor downregulation in the striatum and hypothalamus [41], dysregulation of the apoptosis-controlling gene expression [42] could also contribute to the motor impairment found in these BION SF mice. A less pronounced reduction of grip strength when measured directly in space on ISS [43] implies that return to gravity might exacerbate the microgravity-induced decrease in muscle strength. ...
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Profound effects of spaceflight on the physiology of humans and non-human animals are well-documented but incompletely explored. Current goals to undertake interplanetary missions increase the urgency to learn more about adaptation to prolonged spaceflight and readaptation to Earth-normal conditions, especially with the inclusion of radiation exposures greater than those confronted in traditional, orbital flights. The 30-day-long Bion M-1 biosatellite flight was conducted at a relatively high orbit, exposing the mice to greater doses of radiation in addition to microgravity, a combination of factors relevant to Mars missions. Results of the present studies with mice provide insights into the consequences on brain function of long-duration spaceflight. After landing, mice showed profound deficits in vestibular responses during aerial drop tests. Spaceflown mice displayed reduced grip strength, rotarod performance, and voluntary wheel running, each, which improved gradually but incompletely over the 7-days of post-flight testing. Continuous monitoring in the animals’ home cage activity, in combination with open-field and other tests of motor performance, revealed indices of altered affect, expressed as hyperactivity, potentiated thigmotaxis, and avoidance of open areas which, together, presented a syndrome of persistent anxiety-like behavior. A learned, operant response acquired before spaceflight was retained, whereas the acquisition of a new task was impaired after the flight. We integrate these observations with other results from Bion-M1’s program, identifying deficits in musculoskeletal and cardiovascular systems, as well as in the brain and spinal cord, including altered gene expression patterns and the accompanying neurochemical changes that could underlie our behavioral findings.
... Several treatments have been proposed and used for countering muscle atrophy in humans. Inhibition of a protein called myostatin has shown to result in an increase in muscle mass (Smith et al., 2020). The drug formeterol has been used for counteracting muscle atrophy in mice in spaceflight (Ballerini et al., 2020). ...
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Muscle atrophy is a side effect of several terrestrial diseases which also affects astronauts severely in space missions due to the reduced gravity in spaceflight. An integrative graph-theoretic network-based drug repurposing methodology quantifying the interplay of key gene regulations and protein–protein interactions in muscle atrophy conditions is presented. Transcriptomic datasets from mice in spaceflight from GeneLab have been extensively mined to extract the key genes that cause muscle atrophy in organ muscle tissues such as the thymus, liver, and spleen. Top muscle atrophy gene regulators are selected by Bayesian Markov blanket method and gene–disease knowledge graph is constructed using the scalable precision medicine knowledge engine. A deep graph neural network is trained for predicting links in the network. The top ranked diseases are identified and drugs are selected for repurposing using drug bank resource. A disease drug knowledge graph is constructed and the graph neural network is trained for predicting new drugs. The results are compared with machine learning methods such as random forest, and gradient boosting classifiers. Network measure based methods shows that preferential attachment has good performance for link prediction in both the gene–disease and disease–drug graphs. The receiver operating characteristic curves, and prediction accuracies for each method show that the random walk similarity measure and deep graph neural network outperforms the other methods. Several key target genes identified by the graph neural network are associated with diseases such as cancer, diabetes, and neural disorders. The novel link prediction approach applied to the disease drug knowledge graph identifies the Monoclonal Antibodies drug therapy as suitable candidate for drug repurposing for spaceflight induced microgravity. There are a total of 21 drugs identified as possible candidates for treating muscle atrophy. Graph neural network is a promising deep learning architecture for link prediction from gene–disease, and disease–drug networks.
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Osteogenesis imperfecta (OI) is a collagen‐related bone disorder characterized by fragile osteopenic bone and muscle weakness. We have previously demonstrated that the soluble activin receptor type IIB decoy (sActRIIB) molecule increases muscle mass and improves bone strength in the mild to moderate G610C mouse model of OI. The sActRIIB molecule binds multiple transforming growth factor‐β (TGF‐β) ligands, including myostatin and activin A. Here, we investigate the musculoskeletal effects of inhibiting activin A alone, myostatin alone, or both myostatin and activin A in wildtype (Wt) and heterozygous G610C (+/G610C) mice using specific monoclonal antibodies. Male and female Wt and +/G610C mice were treated twice weekly with intra‐peritoneal injections of monoclonal control antibody (Ctrl‐Ab, Regn1945), anti‐activin A antibody (ActA‐Ab, Regn2476), anti‐myostatin antibody (Mstn‐Ab, Regn647) or both ActA‐Ab and Mstn‐Ab (Combo, Regn2476 and Regn647) from 5 to 16 weeks of age. Prior to euthanasia, whole body composition, metabolism and muscle force generation assessments were performed. Post euthanasia, hindlimb muscles were evaluated for mass, and femurs were evaluated for changes in microarchitecture and biomechanical strength using microCT (μCT) and 3‐point bend analyses. ActA‐Ab treatment minimally impacted the +/G610C musculoskeleton, and was detrimental to bone strength in male +/G610C mice. Mstn‐Ab treatment, as previously reported, resulted in substantial increases in hindlimb muscle weights and overall body weights in Wt and male +/G610C mice, but had minimal skeletal impact in +/G610C mice. Conversely, the Combo treatment out‐performed ActA‐Ab alone or Mstn‐Ab alone, consistently increasing hindlimb muscle and body weights regardless of sex or genotype and improving bone microarchitecture and strength in both male and female +/G610C and Wt mice. Combinatorial inhibition of activin A and myostatin more potently increased muscle mass and bone microarchitecture and strength than either antibody alone, recapturing most of the observed benefits of sActRIIB treatment in +/G610C mice. This article is protected by copyright. All rights reserved.
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Skeletal muscle atrophy is a well-known consequence of spaceflight. Because of the potential significant impact of muscle atrophy and muscle dysfunction on astronauts and to their mission, a thorough understanding of the mechanisms of this atrophy and the development of effective countermeasures is critical. Spaceflight-induced muscle atrophy is similar to atrophy seen in many terrestrial conditions, and therefore our understanding of this form of atrophy may also contribute to the treatment of atrophy in humans on Earth. The unique environmental features humans encounter in space include the weightlessness of microgravity, space radiation, and the distinctive aspects of living in a spacecraft. The disuse and unloading of muscles in microgravity are likely the most significant factors that mediate spaceflight-induced muscle atrophy, and have been extensively studied and reviewed. However, there are numerous other direct and indirect effects on skeletal muscle that may be contributing factors to the muscle atrophy and dysfunction seen as a result of spaceflight. This review offers a novel perspective on the issue of muscle atrophy in space by providing a comprehensive overview of the unique aspects of the spaceflight environment and the various ways in which they can lead to muscle atrophy. We systematically review the potential contributions of these different mechanisms of spaceflight-induced atrophy and include findings from both actual spaceflight and ground-based models of spaceflight in humans, animals, and in vitro studies.
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Interest in space habitation has grown dramatically with planning underway for the first human transit to Mars. Despite a robust history of domestic and international spaceflight research, understanding behavioral adaptation to the space environment for extended durations is scant. Here we report the first detailed behavioral analysis of mice flown in the NASA Rodent Habitat on the International Space Station (ISS). Following 4-day transit from Earth to ISS, video images were acquired on orbit from 16- and 32-week-old female mice. Spaceflown mice engaged in a full range of species-typical behaviors. Physical activity was greater in younger flight mice as compared to identically-housed ground controls, and followed the circadian cycle. Within 7–10 days after launch, younger (but not older), mice began to exhibit distinctive circling or ‘race-tracking’ behavior that evolved into coordinated group activity. Organized group circling behavior unique to spaceflight may represent stereotyped motor behavior, rewarding effects of physical exercise, or vestibular sensation produced via self-motion. Affording mice the opportunity to grab and run in the RH resembles physical activities that the crew participate in routinely. Our approach yields a useful analog for better understanding human responses to spaceflight, providing the opportunity to assess how physical movement influences responses to microgravity.
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Purpose of review: Spinal muscular atrophy (SMA) is a genetic disorder of motor neurons in the anterior horns of the spinal cord and brainstem that results in muscle atrophy and weakness. SMA is an autosomal recessive disease linked to deletions of the SMN1 gene on chromosome 5q. Humans have a duplicate gene (SMN2) whose product can mitigate disease severity, leading to the variability in severity and age of onset of disease, and is therefore a target for drug development. Recent findings: Advances in preclinical and clinical trials have paved the way for novel therapeutic options for SMA patients, including many currently in clinical trials. In 2016, the first treatment for SMA has been approved in the USA, an antisense oligonucleotide that increases full-length protein product derived from SMN2. The approval of a first treatment for SMA and the rapid advances in clinical trials provide the prospect for multiple approaches to disease modification. There are several other promising therapeutics in different stages of development, based on approaches such as neuroprotection, or gene therapy.
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Background: Patients experiencing disuse atrophy report acute loss of skeletal muscle mass which subsequently leads to loss of strength and physical capacity. In such patients, especially the elderly, complete recovery remains a challenge even with improved nutrition and resistance exercise. This study aimed to explore the clinical potential of bimagrumab, a human monoclonal antibody targeting the activin type II receptor, for the recovery of skeletal muscle volume from disuse atrophy using an experimental model of lower extremity immobilization. Methods: In this double-blind, placebo-controlled trial, healthy young men (n = 24; mean age, 24.1 years) were placed in a full-length cast of one of the lower extremities for 2 weeks to induce disuse atrophy. After cast removal, subjects were randomized to receive a single intravenous (i.v.) dose of either bimagrumab 30 mg/kg (n = 15) or placebo (n = 9) and were followed for 12 weeks. Changes in thigh muscle volume (TMV) and inter-muscular adipose tissue (IMAT) and subcutaneous adipose tissue (SCAT) of the thigh, maximum voluntary knee extension strength, and safety were assessed throughout the 12 week study. Results: Casting resulted in an average TMV loss of -4.8% and comparable increases in IMAT and SCAT volumes. Bimagrumab 30 mg/kg i.v. resulted in a rapid increase in TMV at 2 weeks following cast removal and a +5.1% increase above pre-cast levels at 12 weeks. In comparison, TMV returned to pre-cast level at 12 weeks (-0.1%) in the placebo group. The increased adiposity of the casted leg was sustained in the placebo group and decreased substantially in the bimagrumab group at Week 12 (IMAT: -6.6%, SCAT: -3.5%). Knee extension strength decreased by ~25% in the casted leg for all subjects and returned to pre-cast levels within 6 weeks after cast removal in both treatment arms. Bimagrumab was well tolerated with no serious or severe adverse events reported during the study. Conclusions: A single dose of bimagrumab 30 mg/kg i.v. safely accelerated the recovery of TMV and reversal of accumulated IMAT following 2 weeks in a joint-immobilizing cast.
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This Japan Aerospace Exploration Agency project focused on elucidating the impacts of partial gravity (partial g) and microgravity (μg) on mice using newly developed mouse habitat cage units (HCU) that can be installed in the Centrifuge-equipped Biological Experiment Facility in the International Space Station. In the first mission, 12 C57BL/6 J male mice were housed under μg or artificial earth-gravity (1 g). Mouse activity was monitored daily via downlinked videos; μg mice floated inside the HCU, whereas artificial 1 g mice were on their feet on the floor. After 35 days of habitation, all mice were returned to the Earth and processed. Significant decreases were evident in femur bone density and the soleus/gastrocnemius muscle weights of μg mice, whereas artificial 1 g mice maintained the same bone density and muscle weight as mice in the ground control experiment, in which housing conditions in the flight experiment were replicated. These data indicate that these changes were particularly because of gravity. They also present the first evidence that the addition of gravity can prevent decreases in bone density and muscle mass, and that the new platform ‘MARS’ may provide novel insights on the molecular-mechanisms regulating biological processes controlled by partial g/μg.
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Objectives: To assess the effects of bimagrumab on skeletal muscle mass and function in older adults with sarcopenia and mobility limitations. Design: A 24-week, randomized, double-blind, placebo-controlled, parallel-arm, proof-of-concept study. Setting: Five centers in the United States. Participants: Community-dwelling adults (N = 40) aged 65 and older with gait speed between 0.4 and 1.0 m/s over 4 m and an appendicular skeletal muscle index of 7.25 kg/m(2) or less for men and 5.67 kg/m(2) or less for women. Intervention: Intravenous bimagrumab 30 mg/kg (n = 19) or placebo (n = 21). Measurements: Change from baseline in thigh muscle volume (TMV), subcutaneous and intermuscular fat, appendicular and total lean body mass, grip strength, gait speed, and 6-minute walk distance (6MWD). Results: Thirty-two (80%) participants completed the study. TMV increased by Week 2, was sustained throughout the treatment period, and remained above baseline at the end of study in bimagrumab-treated participants, whereas there was no change with placebo treatment (Week 2: 5.15 ± 2.19% vs -0.34 ± 2.59%, P < .001; Week 4: 6.12 ± 2.56% vs 0.16 ± 3.42%, P < .001; Week 8: 8.01 ± 3.70% vs 0.35 ± 3.32%, P < .001; Week 16: 7.72 ± 5.31% vs 0.42 ± 5.14%, P < .001; Week 24: 4.80 ± 5.81% vs -1.01 ± 4.43%, P = .002). Participants with slower walking speed at baseline receiving bimagrumab had clinically meaningful and statistically significantly greater improvements in gait speed (mean 0.15 m/s, P = .009) and 6MWD (mean 82 m, P = .022) than those receiving placebo at Week 16. Adverse events in the bimagrumab group included muscle-related symptoms, acne, and diarrhea, most of which were mild in severity and resolved by the end of study. Conclusion: Treatment with bimagrumab over 16 weeks increased muscle mass and strength in older adults with sarcopenia and improved mobility in those with slow walking speed.
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Risk for premature osteoporosis is a major health concern in astronauts and cosmonauts and the reversibility of the bone lost at the weight-bearing bone sites is not established although it is suspected to take longer than the mission length. The bone three-dimensional structure and strength which could be uniquely affected by weightlessness, is currently unknown. Our objective is to evaluate bone mass, microarchitecture and strength of weight- and non-weight-bearing bone in 13 cosmonauts before and for 12 months after 4-6-month sojourn in the International Space Station (ISS). Standard and advanced evaluations of trabecular and cortical parameters were performed using high-resolution peripheral quantitative computed tomography. In particular, cortical analyses involved determination of the largest common volume of each successive individual scan to improve the precision of cortical porosity and density measurements. Bone resorption and formation serum markers, and markers reflecting osteocyte activity or periosteal metabolism (sclerostin, periostin) were evaluated. At the tibia, in addition to decreased bone mineral densities at cortical and trabecular compartments, a 4% decrease in cortical thickness and a 15% increase in cortical porosity were observed at landing. Cortical size and density subsequently recovered and serum periostin changes were associated with cortical recovery during the year after landing. However tibial cortical porosity or trabecular bone failed to recover, resulting in compromised strength. The radius, preserved at landing, unexpectedly developed post-flight fragility, from 3 months post-landing onwards, particularly in its cortical structure. Remodeling markers, uncoupled in favor of bone resorption at landing, returned to pre-flight values within six months, then declined further to lower than pre-flight values. Our findings highlight the need for specific protective measures not only during, but also after spaceflight, due to continuing uncertainties regarding skeletal recovery long after landing. This article is protected by copyright. All rights reserved.
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The weightless environment during spaceflight induces site-specific bone loss. The 30-day Bion-M1 mission offered a unique opportunity to characterize the skeletal changes after spaceflight and an 8-day recovery period in mature male C57/BL6 mice. In the femur metaphysis, spaceflight decreased the trabecular bone volume (−64% vs. Habitat Control), dramatically increased the bone resorption (+140% vs. Habitat Control) and induced marrow adiposity invasion. At the diaphysis, cortical thinning associated with periosteal resorption was observed. In the Flight animal group, the osteocyte lacunae displayed a reduced volume and a more spherical shape (synchrotron radiation analyses), and empty lacunae were highly increased (+344% vs. Habitat Control). Tissue-level mechanical cortical properties (i.e., hardness and modulus) were locally decreased by spaceflight, whereas the mineral characteristics and collagen maturity were unaffected. In the vertebrae, spaceflight decreased the overall bone volume and altered the modulus in the periphery of the trabecular struts. Despite normalized osteoclastic activity and an increased osteoblast number, bone recovery was not observed 8 days after landing. In conclusion, spaceflight induces osteocyte death, which may trigger bone resorption and result in bone mass and microstructural deterioration. Moreover, osteocyte cell death, lacunae mineralization and fatty marrow, which are hallmarks of ageing, may impede tissue maintenance and repair.
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Mechanical unloading in microgravity during spaceflight is known to cause muscular atrophy, changes in muscle fiber composition, gene expression, and reduction in regenerative muscle growth. Although some limited data exists for long-term effects of microgravity in human muscle, these processes have mostly been studied in rodents for short periods of time. Here we report on how long-term (30-day long) mechanical unloading in microgravity affects murine muscles of the femoral Quadriceps group. To conduct these studies we used muscle tissue from 6 microgravity mice, in comparison to habitat (7), and vivarium (14) ground control mice from the NASA Biospecimen Sharing Program conducted in collaboration with the Institute for Biomedical Problems of the Russian Academy of Sciences, during the Russian Bion M1 biosatellite mission in 2013. Muscle histomorphology from microgravity specimens showed signs of extensive atrophy and regenerative hypoplasia relative to ground controls. Specifically, we observed a two-fold decrease in the number of myonuclei, compared to vivarium and ground controls, and central location of myonuclei, low density of myofibers in the tissue, and of myofibrils within a fiber, as well as fragmentation and swelling of myofibers. Despite obvious atrophy, muscle regeneration nevertheless appeared to have continued after 30 days in microgravity as evidenced by thin and short newly formed myofibers. Many of them, however, showed evidence of apoptotic cells and myofibril degradation, suggesting that long-term unloading in microgravity may affect late stages of myofiber differentiation. Ground asynchronous and vivarium control animals demonstrated normal, well-developed tissue structure with sufficient blood and nerve supply and evidence of regenerative formation of new myofibers free of apoptotic nuclei. Regenerative activity of satellite cells in muscles was observed both in microgravity and ground control groups, using Pax7 and Myogenin immunolocalization, as well as Myogenin expression analysis. In addition, we have detected positive nuclear immunolocalization of c-Jun and c-Myc proteins indicating their sensitivity to changes in gravitational loading in a given model. In summary, long-term spaceflight in microgravity caused significant atrophy and degeneration of the femoral Quadriceps muscle group, and it may interfere with muscle regenerative processes by inducing apoptosis in newly-formed myofibrils during their differentiation phase.
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Mechanical loading is necessary for the development and maintenance of the musculoskeletal system. Removal of loading via microgravity, paralysis, or bed rest leads to rapid loss of muscle mass and function; however, the molecular mechanisms that lead to these changes are largely unknown, particularly for the spaceflight (SF) microgravity environment. Furthermore, few studies have explored these effects on the shoulder, a dynamically stabilized joint with a large range of motion; therefore, we examined the effects of microgravity on mouse shoulder muscles for the 15-d Space Transportation System (STS)-131, 13-d STS-135, and 30-d Bion-M1 missions. Mice from STS missions were euthanized within 4 h after landing, whereas mice from the Bion-M1 mission were euthanized within 14 h after landing. The motion-generating deltoid muscle was more sensitive to microgravity than the joint-stabilizing rotator cuff muscles. Mice from the STS-131 mission exhibited reduced myogenic (Myf5 and -6) and adipogenic (Pparg, Cebpa, and Lep) gene expression, whereas either no change or an increased expression of these genes was observed in mice from the Bion-M1 mission. In summary, muscle responses to microgravity were muscle-type specific, short-duration SF caused dramatic molecular changes to shoulder muscles and responses to reloading upon landing were rapid.-Shen, H., Lim, C., Schwartz, A. G., Andreev-Andrievskiy, A., Deymier, A. C., Thomopoulos, S. Effects of spaceflight on the muscles of the murine shoulder.
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Background: Skeletal muscle is a key mediator of insulin resistance. Bimagrumab, an antibody against activin receptor type II (ActRII), prevents binding of negative muscle regulators, like myostatin, and increases lean mass and decreases fat mass in animal models. Objective: We hypothesized that an improving body composition in insulin resistant individuals could enhance insulin sensitivity. Methods: Sixteen individuals with mean body mass index (BMI) = 29.3 kg/m(2) and insulin resistance, received a single dose of bimagrumab or placebo and were assessed at Week 10 for insulin sensitivity, with hyperinsulinemic euglycemic (H-E) clamp and intravenous glucose tolerance test (IVGTT), and for body composition, with dual energy X-ray absorptiometry (DXA) and Positron emission tomography (PET) scan. Results: Bimagrumab increased lean mass by 2.7% (p < 0.05) and reduced fat mass by 7.9% (p = 0.011) at Week 10 compared to placebo, with a neutral effect on body weight. Bimagrumab reduced HbA1c by 0.21% at Week 18 (p < 0.001) and improved insulin sensitivity by ~20% (using the clamp) to ~40% (using the IVGTT). Conclusion: Taken the observed changes together and in consideration that these occurred without accompanying dietary intervention and without any prescribed regular physical exercise, bimagrumab may offer a novel approach for the treatment of metabolic complications of obesity.