Prolonged space flight‐induced alterations in the structure and function of human skeletal muscle fibres

Abstract

The primary goal of this study was to determine the effects of prolonged space flight (∼180 days) on the structure and function of slow and fast fibres in human skeletal muscle. Biopsies were obtained from the gastrocnemius and soleus muscles of nine International Space Station crew members ∼45 days pre- and on landing day (R+0) post-flight. The main findings were that prolonged weightlessness produced substantial loss of fibre mass, force and power with the hierarchy of the effects being soleus type I > soleus type II > gastrocnemius type I > gastrocnemius type II. Structurally, the quantitatively most important adaptation was fibre atrophy, which averaged 20% in the soleus type I fibres (98 to 79 μm diameter). Atrophy was the main contributor to the loss of peak force (P0), which for the soleus type I fibre declined 35% from 0.86 to 0.56 mN. The percentage decrease in fibre diameter was correlated with the initial pre-flight fibre size (r= 0.87), inversely with the amount of treadmill running (r= 0.68), and was associated with an increase in thin filament density (r= 0.92). The latter correlated with reduced maximal velocity (V0) (r=−0.51), and is likely to have contributed to the 21 and 18% decline in V0 in the soleus and gastrocnemius type I fibres. Peak power was depressed in all fibre types with the greatest loss (∼55%) in the soleus. An obvious conclusion is that the exercise countermeasures employed were incapable of providing the high intensity needed to adequately protect fibre and muscle mass, and that the crew's ability to perform strenuous exercise might be seriously compromised. Our results highlight the need to study new exercise programmes on the ISS that employ high resistance and contractions over a wide range of motion to mimic the range occurring in Earth's 1 g environment.

Full text

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J Physiol 588.18 (2010) pp 3567–3592
3567
Prolonged space flight-induced alterations in the structure
and function of human skeletal muscle fibres
R. H. Fitts1, S. W. Trappe2, D. L. Costill2, P. M. Gallagher2, A. C. Creer2, P. A. Colloton1, J. R. Peters1,
J. G. Romatowski1, J. L. Bain3and D. A. Riley3
1Marquette University, Milwaukee, WI 53201-1881, USA
2Ball State University, Muncie, IN 47306, USA
3Medical College of Wisconsin, Milwaukee, WI 53226, USA
Theprimarygoalofthisstudywastodeterminetheeffectsofprolongedspaceflight(∼180days)
on the structure and function of slow and fast fibres in human skeletal muscle. Biopsies were
obtained from the gastrocnemius and soleus muscles of nine International Space Station crew
members ∼45days pre- and on landing day (R+0) post-flight. The main findings were that
prolonged weightlessness produced substantial loss of fibre mass, force and power with the
hierarchy of the effects being soleus type I > soleus type II > gastrocnemius type I > gastro-
cnemius type II. Structurally, the quantitatively most important adaptation was fibre atrophy,
which averaged 20% in the soleus type I fibres (98 to 79μm diameter). Atrophy was the main
contributor to the loss of peak force (P0), which for the soleus type I fibre declined 35%
from 0.86 to 0.56mN. Thepercentage decrease in fibre diameter was correlated with the initial
pre-flight fibre size (r =0.87), inversely with the amount of treadmill running (r =0.68), and
was associated with an increase in thin filament density (r =0.92). The latter correlated with
reduced maximal velocity (V0) (r =−0.51), and is likely to have contributed to the 21 and 18%
declineinV0inthesoleusandgastrocnemiustypeIfibres.Peakpowerwasdepressedinallfibre
types with the greatest loss (∼55%) in the soleus. An obvious conclusion is that the exercise
countermeasuresemployedwereincapableofprovidingthehighintensityneededtoadequately
protect fibre and muscle mass, and that the crew’s ability to perform strenuous exercise might
beseriouslycompromised.Ourresultshighlighttheneedtostudynewexerciseprogrammeson
the ISS that employ high resistance and contractions over a wide range of motion to mimic the
range occurring in Earth’s 1g environment.
(Resubmitted 15 February 2010; accepted after revision 19 July 2010; first published online 26 July 2010)
Correspondingauthor R. H. Fitts: Marquette University, Department of Biological Sciences, PO Box 1881, Milwaukee,
WI 53201-1881, USA. Email: robert.fitts@marquette.edu
Abbreviations CSA, cross-sectional area; FL, fibre length; ISS, International Space Station; ktr, rate constant of tension
redevelopment; P0, peak force; V0, maximal unloaded shortening velocity.
Introduction
The goals of the international space community are to
conduct long-termed manned missions beyond the low
earth orbit of the International Space Station (ISS). First,
a number of issues regarding the deleterious effects of
microgravity on human biology need to be addressed and
resolved (Fitts et al. 2000; Trappe et al. 2009). It is clear
fromthelast40yearsofspaceresearch,particularlystudies
conducted on the Skylab and MIR space stations and on
mission STS-78 of the Space Shuttle Columbia (with the
Life and Microgravity Spacelab, LMS), that limb skeletal
muscleisparticularlysusceptibletomicrogravity-induced
deteriorationinbothstructureandfunction(Convertino,
1990; Fitts et al. 2000; Fitts et al. 2001). A consistent
observationissignificantatrophyofbothupperandlower
leg muscles with the response occurring more rapidly in
thetricepssuraemusclegroup(ankleplantarflexors)than
the anterior tibial group (ankle dorsal flexors) (Fitts et al.
2000). The primary cause of the decline in muscle mass
appears to be the unloading of the skeletal and muscular
systems rather than reduced activation. Support for this
comes from the work of Edgerton et al. (2001) who
found the total EMG activity of the tibialis anterior and
soleus muscles of four crew members during the 17-day
LMS space flight to be increased compared to pre- and
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3568R. H. Fitts and others
J Physiol 588.18
post-flightvalues.Theauthorsconcludedthatspaceflight
on Shuttle missions is a model not just of space flight but
rather microgravity plus the programmed work schedule.
Despite the high EMG activity, the cross-sectional areas
(CSAs) of the slow type I and fast type IIa fibres of the
soleus muscles of the four crew members were on average
15and26%smallerpost-comparedtopre-flight(Widrick
et al. 1999).
The composite data from Skylab, MIR and Shuttle
flights suggest that the loss of limb muscle mass is
exponential with the duration of flight, and that a micro-
gravity steady state may be reached by approximately
180days (Fitts et al. 2000). The loss in muscle force
primarilyreflectsthedeclineinmass.Consequently,when
single fibre force is expressed relative to cross-sectional
area, there is little difference between pre- and post-flight
values(Widricketal.1999;Fittsetal.2000).Inadditionto
the decline in muscle mass and peak force, crew members
showed a depressed ability to generate power that was
generally greater than the loss of force (Widrick et al.
1999;Fittsetal.2000).Forexample,after31daysinspace,
lower limb extensor force declined by 11%, while peak
power was depressed by 54% (Antonutto et al. 1999). The
latter was greater than the loss in single-fibre power after
the 17-day LMS flight suggesting that factors other than
atrophy contributed to the decline (Widrick et al. 1999;
Fitts et al. 2000). Following the LMS flight, we found that
the decline in peak power was partially protected by an
increased maximal velocity (V0) of both slow and fast
fibres such that the velocity obtained at peak power was
higher post-flight (Widrick et al. 1999). The elevated fibre
V0was associated with and likely caused by an increase in
myofilamentlatticespacingthatresultedfromareduction
inthinfilamentdensity.Itisunknownwhetherornotthis
adaptation persists with prolonged space flight or reflects
a transient response to short duration flight.
Ahallmarkofspaceflightisthatconsiderablevariability
in the extent of muscle atrophy and functional loss exists
among crew members. For example, the crew members in
this study showed calf muscle atrophy ranging from 1 to
>20% and loss of maximal voluntary contractile force of
thecalffrom7to20%(Trappeetal.2009).Similarly,Zange
et al. (1997) observed muscle mass losses with 6months
in space to vary from 6 to 20%. Following the 17-day
LMS flight, two crew members showed 2–3 times the
reduction in peak force (mN) noted for the other two
(Widrick et al. 1999). Besides mass and force, variability
was also observed for Ca2+sensitivity where the free Ca2+
requiredforhalf-maximalactivationofsoleustypeIfibres
post-flight ranged from no difference to 0.31μmol more
free Ca2+.
The primary goal of this study was to use single,
chemically skinned muscle fibre segments to determine
the cellular effects of prolonged (∼180days) space
flight aboard the International Space Station (ISS). An
additional goal was to determine the extent to which
the observed functional changes were fibre and muscle
specific, and whether or not they could be explained by
structural alterations. Changes in structure and function
were to the extent possible related to differences in the
type and amount of countermeasure exercise. To allow
for scientific comparisons between the single fibre results
described here and our recently published whole muscle
data on the same subjects (Trappe et al. 2009), the letter
code used for a given crew member was the same in both
publications. The exercise countermeasure performed by
each crew member was presented in detail in Trappe et al.
(2009). The results were also compared to the known cell
changes following short duration space flight.
Methods
Flight and subjects
The 10 crew members, five American astronauts and
five Russian cosmonauts, who participated in this study
flew aboard the International Space Station (ISS) from
Increments 5 to 11 (2002–2005). All flights except for the
first(Increment5)originatedandlandedinRussiaaboard
the Russian Soyuz spacecraft. The crew of Increment 5
were ferried to and from the ISS on the Space Shuttle,
which lifted off and landed at Kennedy Space Center. The
post-flight muscle samples for one crew member were
damaged during shipment from Russia to the USA, and
thus the data for this subject were not included. The
subjects (n=9) age, height, weight and days in space
were45±2years,176±2cm,81±3kg,and177±4days
(range=161–192days), respectively.
Prior to volunteering to participate in this study, all
crew members were briefed on the project objectives
and testing procedures by a member of the research
team. Crew members were informed of the risks and
benefits of the research and gave their written consent in
accordancewiththeHumanSubjectsInstitutionalReview
Boards at Marquette University, Ball State University,
The Medical College of Wisconsin, and the National
Aeronautics and Space Administration (NASA; Johnson
Space Center). This study was conducted in accordance
with the Declaration of Helsinki.
Pre- and in-flight exercise and nutritional profile
The pre- and in-flight exercise programmes of each crew
member have been published elsewhere by our research
team (Trappe et al. 2009). The crew members had access
to a treadmill (Treadmill Vibration Isolation System;
TVIS), two types of bicycle ergometers (Cycle Ergometer
with Vibration Isolation System (CEVIS) and Velo, a
Russian exercise device), and a resistive exercise device
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J Physiol 588.18
Prolonged space flight and human muscle3569
Table 1. Summary of aerobic and resistance exercise performed while on the ISS
Cycle ergometer (CEVIS)Treadmill (TVIS) Resistance exercise (iRED)
Time (min week−1)Workload (W)Time (min week−1)Speed (mph) ExercisesFrequencySets/reps
138 ± 26
Range:
Little – 296
126 ± 10
Range:
102 – 150
146 ± 32
Range:
64 – 312
3.2 ± 0.5
Range:
2.1 – 5.5
Squats
Heel raises
Dead lifts
3–6 days week−1
3–6 days week−1
3–6 days week−1
12–20
12–20
12–20
For more detailed information about the exercise prescription performed while on the ISS and individual aerobic
resistance exercise data profiles for each crew member, see Trappe et al. (2009).
(Interim Resistive Exercise Device; iRED). The treadmill
could be used in a passive (subject driven) or active
(motorized) mode of operation. The exercise counter-
measure programme was individually structured to allow
for personal preference with guidance from staff within
NASA and the Russian Space Agencies. A summary of the
in-flight exercise is shown in Table1. For more detailed
information about the exercise prescription performed
while on the ISS and individual aerobic and resistance
exercise data profiles for each crew member see Trappe
et al. (2009). The exercise profiles were determined from
crew member logbooks and from downloaded analog
data from the treadmill and cycle ergometer (Trappe
et al. 2009). The in-flight diet was designed to meet the
nutritional requirements for ISS missions as established
byNASAandtheRussianSpaceAgencies(Smith&Zwart,
2008). The nutrient content of the pre- and post-flight
foods was calculated using the Nutrient Data System for
Research (Schakel et al. 1988).
Muscle biopsy
A muscle biopsy (Bergstrom, 1962) of ∼80mg was
obtained from the mid-belly of the lateral head of
the gastrocnemius and soleus muscles of each crew
member prior to launch (L-55±2) and on landing day
(R+0) as described previously (Trappe et al. 2009). The
post-flight biopsy was performed mid-to-late afternoon
approximately6–8hafterlanding.Thepost-flightactivity
between landing and the biopsy was kept to a minimum,
and during that time the crew members performed only
light ambulatory activities.
Each biopsy sample was placed on saline-soaked gauze
and divided longitudinally into several portions for sub-
sequent structural and functional analyses exactly as
described previously (Widrick et al. 1999). Two portions
of each biopsy were placed in small vials containing cold
(4◦C) skinning solution (125mM potassium propionate,
20mM imidazole, 2mM EGTA, 4mM ATP, 1mM MgCl2,
and 50% glycerol v/v, pH 7.0), and stored overnight at
4◦C. The next day, the vials were packaged surrounded by
frozen,watericebottlesintwoboxeswitheachcontaining
two vials (one soleus and one gastrocnemius sample),
handcarriedbacktoBallStateandMarquetteUniversities.
Upon arrival, the bundles were placed in fresh skinning
solutionandstoredat−20◦Cforupto4weeks.Allcontra-
ctile measurements on fibres from a given muscle were
performed within 4 weeks of the initial bundle isolation.
Athirdportionofeachbiopsywaspinnedatamildstretch
and immersion fixed in a 0.1 M cacodylate buffer (pH 7.2)
containing 4% glutaraldehyde and 2% paraformaldehyde
with 5mM CaCl2. This sample was shipped overnight at
4◦C to the Medical College of Wisconsin for osmium
post-fixation and embedding for electron microscopy as
previously described (Riley et al. 1998). The fourth and
fifth portions were frozen in liquid nitrogen and shipped
in a liquid nitrogen dry shipper to Marquette University.
Solutions
The composition of the relaxing (pCa 9.0) and activating
(pCa 4.5) solutions were derived with an iterative
computer program (Fabiato & Fabiato, 1979) using the
stability constantsadjusted for temperature,pH and ionic
strength (Godt & Lindley, 1982). All solutions contained
(in mM): 20 imidazole, 7 EGTA, 14.5 creatine phosphate,
4 free ATP, and 1 free Mg2+. Calcium was added as CaCl2,
and ATP as a disodium salt. Each solution had an ionic
strength of 180mM, which was controlled by varying the
amount of KCl added. KOH was used to adjust the pH
of the solution to 7.0. To prevent an increase in ADP
or decline in ATP, we changed the activating solution
after every two contractions. The activating and relaxing
solutions were made fresh each week and stored at 4◦C.
Single fibre preparation
This study involved the isolation and study of 1900 fibres
with experiments conducted in the labs of Dr Fitts at
Marquette and Dr Trappe at Ball State Universities. The
procedures described here for the isolation and study of
individual fibres were the same in both labs. While the
single fibre systems used were similar, the equipment was
not identical. Since the results obtained by each lab for all
variables studied were similar, the data were pooled and
presented here as one data set.
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3570 R. H. Fitts and others
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Single fibres were isolated and studied as described
previously and briefly reviewed here (Widrick et al. 1999;
Fitts et al. 2007). On the day of an experiment a muscle
bundlewasremovedfromtheskinningsolutionandtrans-
ferred to a dissection chamber containing pH 7.0 relaxing
solution (4◦C). An individual fibre was gently isolated
from the bundle, transferred to an ∼1ml glass-bottomed
chambermilledinastainlesssteelplate.Whilesubmerged
under relaxing solution (pH 7.0, 15◦C), the ends of the
fibre were carefully mounted and attached between a
force transducer (Cambridge model 400A; Cambridge
Technology, Watertown, MA, USA) and servo-controlled
direct-current position motor (Cambridge model 300B,
Cambridge Technology). The position and speed of
the motor was controlled by custom-designed software
running on a microcomputer interfaced with a National
Instrumentsdataacquisitionboard(NI-DAQ).Todisrupt
anyremainingintactmembranes,thefibrewassubmerged
into a relaxing solution containing 0.5% Brij 58 for 30s
after which the fibre bath was exchanged twice with
relaxing solution.
The experimental chamber was mounted on the stage
ofaninvertedmicroscope.Sarcomerelengthwasadjusted
to 2.5μm using an eyepiece micrometer (800×), and the
length of the fibre (FL) was recorded. A digital photo (Pro
IDEOCVC-140camera)wastakenofthefibrewhileitwas
brieflysuspendedinair.Fibrediameterwasdeterminedat
threepointsalongthelengthofthefibreusingScionImage
software,andfibreCSAcalculatedfromthemeandiameter
measurement, assuming the fibre forms a circular cross
section when suspended in air (Metzger & Moss,
1987).
Experimental procedures
Fibres exhibiting non-uniform sarcomere lengths or
regions of tearing were not studied (Moss, 1979).
Additionally, data for a given fibre were not included
if peak isometric force (P0) declined by >15% or fibre
compliance(determinedfromthey-axisinterceptofslack
test) exceeded 10% (Trappe et al. 2004). For most fibres,
P0declined <10% from the beginning to the end of the
experiment.ContractilefunctionofindividualfasttypeII
and slow type I fibres was determined exactly as described
previously for our 17-day microgravity study (Widrick
et al. 1999). Briefly, the fibre was maximally activated
in pCa 4.5 solution, allowed to reach peak isometric
force (P0), and slacked to a predetermined length, which
caused tension to drop to zero. The time it took the fibre
to take up the slack and initiate the redevelopment of
tension was measured. The fibre was then returned to
relaxing solution (15◦C) and re-extended to its original
fibrelength.Eachfibrewassubjectedtofivedifferentslack
steps and fibre V0(FLs−1) determined from the slope
of the least squares regression line of the plot of slack
distance versus the time required for the redevelopment
of force. Slack length changes never exceeded 20% of fibre
length.
The rate constant of tension redevelopment (ktr) was
determined using the slack–unslack procedure (Metzger
& Moss, 1990). To prevent sarcomere non-uniformity
during tension redevelopment, Metzger & Moss (1990)
usedalasertoclampthesarcomeresat2.5μm.Theclamp
procedure is time consuming and not practical when
hundreds of fibres are studied. In preliminary studies, we
determinedthatthektroftheslowtypeIfibrewasidentical
with and without a laser clamp, while the fast type II fibre
showed a significantly lower ktrin the absence of a laser
clamp (Fitzsimons et al. 2001). Thus, we determined the
ktrin slow but not fast fibres. The measurement requires
activationofthefibreinpCa4.5andfollowingattainment
of steady tension a 400μm slack, a 40ms delay, and then
re-extension to the original FL. Re-extension dissociates
the cross-bridges, and tension redevelopment was best
fitted with a first-order exponential equation where the
rateconstantk isktrandthoughttoreflecttheratelimiting
step in the generation of the high force state (Metzger &
Moss, 1990).
FollowingthedeterminationofV0andktr,isotonicload
clamps were employed to measure force–velocity–power
parameters. For each fibre, the force (as a percentage of
peak force) and the corresponding shortening velocity
for 15 force–velocity data points were fitted to the Hill
equation with the use of an iterative non-linear
curve-fittingprocedure
algorithm), and maximal shortening velocity (Vmax) and
the a/P0 ratio determined (Widrick et al. 1998). Peak
fibre power was calculated with the fitted parameters of
the force–velocity curve and P0 (Widrick et al. 1998).
Composite force–velocity and force–power curves were
constructed by summating velocities or power values
from 0 to 100% of P0in increments of 1%.
In a subset of fibres (see Table11 for the n for each
fibre type), force–pCa relationships were determined by
activating the fibres in a series of solutions with calcium
concentrations ranging from pCa 6.8 to 4.5 exactly as
described previously (Widrick et al. 1999). Hill plots
were fitted to the data and the activation threshold, the
one-half maximal activation (pCa50), and slope of the
force–calcium relationship below (n2) and above (n1)
pCa50determined (Widrick et al. 1999). Fibre stiffness
or the elastic modulus (E0) was measured by oscillating
the position motor at 1.5 kHz at an amplitude of 0.05%
of FL both before (relaxing solution pCa 9.0), and
during the measurement of peak force at the various
pCa values. The elastic modulus E0 at each pCa was
calculated from the equation E0=(?force in activating
solution−?force in relaxing solution/?length) (fibre
length/fibre cross-sectional area).
(Marquardt–Levenberg
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Prolonged space flight and human muscle3571
SDS gel analysis of actin and myosin composition
After the contractile tests, the fibre was removed from
the experimental set-up and solubilized in 10μl of
sodium dodecyl sulfate (SDS) sample buffer (6mgml−1
EDTA, 0.06 M tris(hydroxymethyl)aminomethane, 1%
SDS, 2mgml−1Bromophenol Blue, 15% glycerol, 5%
β-mercaptoethanol), and stored at −80◦C. Fibre types
wereidentifiedbythemyosinheavychain(MHC)isoform
pattern using 5% polyacrylamide gels as slow type I or
fast type II. Fibres containing both slow and fast myo-
sin (hybrid fibres) were not included in the analysis.
Myosin (heavy and light chains), actin, tropomyosin and
troponinprofilesweredeterminedby12%polyacrylamide
gel analysis (Widrick et al. 1997). For type I fibres,
a computer-based image analysis system and software
(LabWorks; UVP Inc., Upland, CA, USA) were used to
quantify the relative density of the MHC and actin bands
on the 12% gels. For Fig.14B, actin/myosin ratio (actin
band intensity/slow myosin band intensity) was plotted
versus thin filament number per square micrometre for
subjects A, C, D, E, F, G, H and I. For each subject, the
actin/myosinratiowasdeterminedonanaverageof26±4
(pre-flight) and 30±6 (post-flight) fibres, and the thin
filament density on five pre- and five post-flight fibres
using electron microscopy.
Analysis of thin filament density by electron
microscopy
Our previous studies demonstrated that in normal rat
and human soleus muscle fibres, thin filaments varied in
length, and during 17-day spaceflight and bedrest, the
percentages of short thin filaments increased (Riley et al.
1998, 2000, 2005). In cross-sectioned sarcomeres, thin
filamentdensitywashighestintheIbandandfelloffinthe
A bandbecausethe shortfilamentsarisingfromtheZ line
were not long enough to reach the A band in a sarcomere
at 2.5μm length. For the present 180-day spaceflight
muscles,aqualitativeinspectionofthinfilamentnumbers
in the I band near the Z line and within the A band (over-
lap A) where thin filaments first overlap thick filaments
indicated that thin filaments were not missing as expected
from 17day flight data but appeared more abundant,
pointingtoincreasedthinfilamentlength.Furtherintothe
AbandnearertheMline,thinfilamentnumberdecreased,
which made sense because only the longest thin filaments
(∼1.27nm) could reach this far. Thus, to detect increased
thinfilamentdensityduetoincreasedthinfilamentlength,
thin filament densities were quantified near the M line in
cross sections of sarcomeres of slow muscle fibres in the
pre- and post-flight biopsy bundles from the solei of each
subject.Theconceptsofthinfilamentlength,thinfilament
density and location of near the M line measurement site
are illustrated diagrammatically in Fig.16.
Cross sections (70nm) of the epoxy-embedded muscle
bundles were cut, contrasted with uranyl acetate and
lead citrate and examined and imaged in a JEOL 100
CXII electron microscope (EM). The near M line regions
of thick and thin filaments of slow fibres were imaged,
and the EM negatives were scanned for computerized
morphometrical analysis using MetaMorph 5.2 software.
As conducted previously to achieve adequate statistical
power, five slow fibres were sampled per soleus muscle
per time point for a total of 90 fibres (Riley et al. 2000,
2005). Group averages are reported as the mean± S.E.M.
The sarcomere length varied in the aldehyde fixed fibres,
and it is known that myofilament density is directly
related to sarcomere length (Riley et al. 2000, 2005).
To normalize for sarcomere length differences among
fibres, thick filament spacing was adjusted to 31.3nm
(2.5μm sarcomere length). After normalization, the
average thick filament density of the pre-flight fibres
(999±21filamentsμm−2) was comparable (P =0.07) to
that of the post-flight fibres (945±19μm2), confirming
standardization. Measurement of thick and thin filament
densities was accomplished by counting the numbers
of each filament type in a 0.0056μm2grid square at
×201,000 magnification on the computer screen using
Gunderson’s rules for sampling (Riley et al. 2000, 2005).
Fornon-biasedsamplingofthickandthinfilamentcounts,
the grid squares were positioned at random over the A
band regions of thick and thin filament overlap within
an estimated 100–300nm of the M band in central myo-
fibrils (Riley et al. 2002). The position of the sampling
square was fine adjusted to insure that the corralled thick
andthinfilamentsappearsasdots,indicatingcrosssection
orientation.
Statistical analysis
Tominimizeoperatorbias,themorphologicalandphysio-
logical measurements were completed independently
before the data were assembled for each subject to assess
structural and functional correlations. For the functional
studies,thefibresstudiedforeachcrewmemberforagiven
parameter were aggregated to obtain pre- and post-flight
means.Thesedatawereanalysedusingaone-wayANOVA
with Tukey’s post hoc test. Group pre- and post-flight
means (high versus low treadmill, and all crew members
combined)wereanalysedwithStudent’st testforunpaired
data. For morphological quantification, the pre- and
post-flight group means were compared by subject paired
t test. The thin filament densities of post-flight soleus
fibres were compared with the control pre-flight fibres
for each crew member using a two-tailed, unpaired t test
analysisoffiveslowfibrespertimepoint.Whencorrelating
thinfilamentdensitywithV0,theindividualmeansofthin
filamentdensitiesforthepre-andpost-flightsampleswere
compared with the V0means for slow fibres isolated from
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3572 R. H. Fitts and others
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Table 2. Diameter and cross sectional area of the soleus and gastrocnemius slow type I fibre pre- and post-flight
Sol Type IGastrocnemius Type I
DiameterCSA DiameterCSA
Crew member
n
(μm)(%)(μm2)(%)
n
(μm)(%)(μm2) (%)
A
Pre-flight
Post-flight
70
45
107 ± 2
58 ± 1∗
9277 ± 416
2663 ± 74∗
56
39
72 ± 2
58 ± 1∗
4150 ± 186
2682 ± 97∗
5428 81 65
B
Pre-flight
Post-flight
39
29
71 ± 2
75 ± 2
4002 ± 193
4461 ± 204106 111
C
Pre-flight
Post-flight
48
47
105 ± 2
83 ± 1∗
8722 ± 316
5535 ± 183∗
10
14
99 ± 3
90 ± 5
7802 ± 443
6597 ± 741 7963 9185
D
Pre-flight
Post-flight
52
49
99 ± 1
78 ± 2∗
7817 ± 223
4822 ± 196∗
32
24
98 ± 2
74 ± 2∗
7627 ± 396
4301 ± 195∗
7962 76 56
E
Pre-flight
Post-flight
45
64
102 ± 3
87 ± 1∗
8358 ± 473
6045 ± 164∗
32
32
100 ± 4
88 ± 1∗
8185 ± 574
6060 ± 170∗
85 72 8874
F
Pre-flight
Post-flight
41
28
121 ± 3
59 ± 1∗
11756 ± 513
2798 ± 120∗
53
13
85 ± 2
69 ± 2∗
5830 ± 245
3732 ± 161∗
49 248164
G
Pre-flight
Post-flight
55
56
97 ± 1
85 ± 1∗
7391 ± 176
5756 ± 208∗
44
27
78 ± 1
67 ± 2∗
4807 ± 118
3556 ± 157∗
88788674
H
Pre-flight
Post-flight
104
71
89 ± 1
77 ± 1∗
6316 ± 131
4676 ± 118∗
42
13
80 ± 1
68 ± 2∗
5027 ± 160
3694 ± 221∗
8774 8573
I
Pre-flight
Post-flight
High treadmill
Pre-flight
Post-flight
Low Treadmill
Pre-flight
Post-flight
All crew members
Pre-flight
Post-flight
86
69
97 ± 1
91 ± 2∗
7522 ± 184
6661 ± 231∗
36
24
65 ± 2
63 ± 1
3381 ± 199
3177 ± 8894 89 9794
243
220
90 ± 1
82 ± 1∗
6517 ± 936
5235 ± 391
118
72
85 ± 1
76 ± 1∗
6006 ± 1091
4437 ± 81392 ± 584 ± 986 ± 174 ± 0
297
238
104 ± 1
77 ± 1∗
9019 ± 753
4496 ± 778∗
187
114
80 ± 1
68 ± 1∗
5758 ± 892
4098 ± 68171 ± 9 53 ± 1285 ± 473 ± 7
540
458
98 ± 1
79 ± 1∗
7907 ± 705
4824 ± 459∗
305
186
82 ± 1
71 ± 1∗
5851 ± 643
4225 ± 490†
80 ± 667 ± 986 ± 2 76 ± 5
High treadmill group subjects B, E, G and H, and low treadmill group subjects A, C, D, F and I; values are means ± S.E.M.; n, no. of fibres
studied. P0, peak isometric force; %, percentage of pre-flight value; V0, maximal shortening velocity; FL, fibre length.∗Significantly
different from pre-flight value, P < 0.05.
the same biopsies. Statistical significance was accepted at
P <0.05. All data are presented as means± S.E.M.
Results
For the determination of soleus fibre diameter, peak force
(P0),andmaximalunloadedshorteningvelocity(V0),600
pre-flight(540typeIand60typeII,atypeI/typeIIratioof
90%),and542post-flight(458typeIand84typeII,atype
I/typeIIratioof85%)fibreswereanalysed.Forthesesame
parameters,thegastrocnemiusanalysisincludedthestudy
of433pre-flight(305typeIand128typeII,atypeI/typeII
ratioof70%),and325post-flight(186typeIand139type
II, a type I/type II ratio of 57%) fibres. The force–velocity
andtheforce–pCarelationshipsweredeterminedonfewer
fibres,asfollowingtheV0determinationonebutnotboth
of these relationships was determined. The main findings
are described in the following paragraphs.
Fibre atrophy and peak force
Pre-andpost-flightdiametersandCSAfortheslowtypeI
fibresofthesoleusandgastrocnemiusareshowninTable2
and peak force (mN and kNm−2) in Tables3 (soleus)
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Prolonged space flight and human muscle3573
Table 3. Peak force and maximal shortening velocity of the soleus slow type I fibre pre-
and post-flight
Po
Vo
Crew member
n
(mN) (%)(kN m−2)(FL s−1) (%)
A
Pre-flight
Post-flight
70
45
0.86 ± 0.04
0.38 ± 0.01∗
97 ± 2
144 ± 4∗
0.90 ± 0.04
0.73 ± 0.04∗
44 81
B
Pre-flight
Post-flight
39
29
0.48 ± 0.02
0.43 ± 0.02
124 ± 4
97 ± 3∗
1.13 ± 0.16
0.80 ± 0.03
90
71
C
Pre-flight
Post-flight
48
47
0.97 ± 0.04
0.55 ± 0.02∗
113 ± 3
102 ± 4
0.60 ± 0.02
0.51 ± 0.03∗
5785
D
Pre-flight
Post-flight
52
49
0.96 ± 0.03
0.55 ± 0.02∗
123 ± 3
116 ± 3
0.93 ± 0.05
0.81 ± 0.04 57 87
E
Pre-flight
Post-flight
45
64
0.78 ± 0.03
0.66 ± 0.02∗
97 ± 3
111 ± 2∗
0.80 ± 0.05
0.83 ± 0.0485104
F
Pre-flight
Post-flight
41
28
1.29 ± 0.05
0.39 ± 0.01∗
114 ± 4
143 ± 4∗
0.96 ± 0.06
0.53 ± 0.03∗
30 56
G
Pre-flight
Post-flight
55
56
0.86 ± 0.03
0.63 ± 0.03∗
118 ± 4
109 ± 3+
0.85 ± 0.04
0.67 ± 0.03∗
7379
H
Pre-flight
Post-flight
104
71
0.74 ± 0.02
0.47 ± 0.02∗
118 ± 2
104 ± 4∗
0.84 ± 0.03
0.62 ± 0.02∗
6474
I
Pre-flight
Post-flight
High treadmill
Pre-flight
Post-flight
Low treadmill
Pre-flight
Post-flight
All crew members
Pre-flight
Post-flight
86
69
0.91 ± 0.02
0.74 ± 0.03∗
124 ± 3
113 ± 3∗
0.75 ± 0.03
0.58 ± 0.03∗
8177
243
220
0.73 ± 0.01
0.56 ± 0.01∗
115 ± 2
106 ± 2∗
0.88 ± 0.03
0.72 ± 0.02∗
77 81
297
238
0.97 ± 0.02
0.56 ± 0.01∗
114 ± 1
121 ± 2∗
0.83 ± 0.02
0.64 ± 0.02∗
5877
540
458
0.86 ± 0.01
0.56 ± 0.01∗
115 ± 1
114 ± 1
0.85 ± 0.02
0.68 ± 0.01∗
65 80
High treadmill group subjects B, E, G and H, and low treadmill group subjects A, C, D, F and
I; values are means ± S.E.M.; n, no. of fibres studied. P0, peak isometric force; %, percentage
of pre-flight value; V0, maximal shortening velocity; FL, fibre length.∗Significantly different
from pre-flight value, P < 0.05. For subject C the n for Vowas 35 and 31 for the pre- and
post-flight sample.
and 4 (gastrocnemius). Corresponding fast fibre data are
shown in Tables5 and 6. In all but the fast gastrocnemius
fibres, prolonged space flight elicited significant atrophy
(as determined from fibre diameter and CSA) and decline
in peak force (mN) with the degree of change soleus
type I > soleus type II > gastrocnemius type I fibres.
Considerablevariabilitybetweencrewmembersexistedin
both the degree of fibre atrophy and the loss of peak force
(Tables2–4). For example, crew member B showed no
soleus type I fibre atrophy, and only a modest 10% loss in
fibre force, whereas for crew member F, soleus type I fibre
size and force were reduced by 51 and 70%, respectively
(Tables2 and 3). The decline in peak force was primarily
caused by fibre atrophy as the crew average for soleus
fibreforceexpressedinkNm−2wasnotalteredpre-versus
post-flight, while absolute force in mN declined by 35%
(Table3). For crew members A and F, soleus fibre atrophy
must have exceeded the contractile filament loss as fibre
force per CSA significantly increased (Table3). For the
gastrocnemiusmuscle,noneofthecrewmembersshowed
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3574R. H. Fitts and others
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Table 4. Peak force and maximal shortening velocity of the gastrocnemius slow type I
fibre pre- and post-flight
Po
Vo
Crew member
n
(mN)(%)(kN m−2)(FL s−1)(%)
A
Pre-flight
Post-flight
56
39
0.47 ± 0.02
0.38 ± 0.01∗
116 ± 3
144 ± 4∗
0.86 ± 0.04
0.71 ± 0.05∗
8182
C
Pre-flight
Post-flight
10
14
0.78 ± 0.05
0.59 ± 0.05∗
100 ± 3
92 ± 5
0.67 ± 0.03
0.59 ± 0.03 76 89
D
Pre-flight
Post-flight
32
24
0.83 ± 0.03
0.46 ± 0.02∗
113 ± 3
118 ± 4
0.95 ± 0.06
0.85 ± 0.055590
E
Pre-flight
Post-flight
32
32
0.82 ± 0.04
0.67 ± 0.02∗
107 ± 5
112 ± 4
1.05 ± 0.06
0.91 ± 0.07 8287
F
Pre-flight
Post-flight
53
13
0.73 ± 0.08
0.45 ± 0.09
136 ± 20
121 ± 5
0.83 ± 0.05
0.65 ± 0.06 6278
G
Pre-flight
Post-flight
44
27
0.53 ± 0.01
0.35 ± 0.02∗
114 ± 4
103 ± 9
0.80 ± 0.05
0.59 ± 0.04∗
6674
H
Pre-flight
Post-flight
42
13
0.56 ± 0.01
0.43 ± 0.04∗
114 ± 3
118 ± 10
0.87 ± 0.04
0.58 ± 0.04∗
7770
I
Pre-flight
Post-flight
High treadmill
Pre-flight
Post-flight
Low treadmill
Pre-flight
Post-flight
All crew members
Pre-flight
Post-flight
36
24
0.37 ± 0.02
0.39 ± 0.02
120 ± 8
125 ± 5
0.80 ± 0.04
0.67 ± 0.04∗
105
84
118
72
0.62 ± 0.02
0.50 ± 0.02∗
112 ± 2
109 ± 4
0.89 ± 0.03
0.73 ± 0.04∗
81 82
187
114
0.60 ± 0.03
0.43 ± 0.01∗
121 ± 6
125 ± 3
0.85 ± 0.02
0.71 ± 0.02∗
7284
305
186
0.61 ± 0.02
0.46 ± 0.01∗
118 ± 4
119 ± 2
0.86 ± 0.02
0.72 ± 0.02∗
75 83
High treadmill group subjects B, E, G and H, and low treadmill group subjects A,
C, D, F and I; values are means ± S.E.M.; n, no. of fibres studied. P0, peak isometric
force; %, percentage of pre-flight value; V0, maximal shortening velocity; FL, fibre
length.∗Significantly different from pre-flight value, P < 0.05.
a significant decrease in type I fibre force expressed as
kNm−2,whileallbutsubjectIshoweddeclinesinabsolute
force that exceeded 18% (Table4).
Thepercentagechange(pre-topost-flight)inthemean
soleustypeIfibrediameterforeachcrewmembershowed
a significant correlation with thepercentage change in the
pre- to post-flight soleus muscle volume determined by
MRI (Fig.1). The mean space flight-induced decline in
soleus type I fibre CSA was 33%, a value that agrees well
withCSAdeterminationsmadeonhistochemicallystained
muscle fibre bundles (Fig.2). In the soleus, the extent of
typeIfibreatrophytendedtobeassociatedwithanincrease
in the number of fast type II fibres post-flight. Thus, crew
memberswiththehighestpercentagedeclineinmeanfibre
diametershowedthegreatestincreaseinthenumberoffast
type II fibres (Fig.3).
The volume of treadmill use appeared to influence the
muscle atrophy response while on the ISS for 6months.
As a result, we subdivided the crew members into those
running 200minweek−1or more (high treadmill) from
those running less than 100minweek−1(low treadmill).
The weekly treadmill running average for each crew
memberhasbeenpublishedelsewhere(Trappeetal.2009).
The high treadmill group showed significantly less space
flight-induced atrophy and loss of force in the soleus slow
type I and fast type II fibres (Tables2, 3 and 5), while
for the gastrocnemius no differences between groups for
these parameters was observed for type I (Tables2 and
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J Physiol 588.18
Prolonged space flight and human muscle3575
Table 5. Diameter, cross sectional area, peak force, and maximal shortening velocity of the soleus fast type II fibre pre-
and post-flight
Diameter CSA
Po
Vo
Condition
n
(μm)(%)(μm2)(%)(mN)(%)(kN m−2) (FL s−1)(%)
High treadmill
Pre-flight
Post-flight
Low treadmill
Pre-flight
Post-flight
All crew members
Pre-flight
Post-flight
37
24
90 ± 2
87 ± 3
6548 ± 296
6170 ± 490
0.82 ± 0.04
0.80 ± 0.07
129 ± 3
130 ± 6
2.62 ± 0.20
2.52 ± 0.2697 94 9896
23
60
105 ± 5
77 ± 2∗
9054 ± 845
4818 ± 296∗
1.20 ± 0.09
0.68 ± 0.03∗
139 ± 5
144 ± 3
2.74 ± 0.25
3.10 ± 0.1773 5357113
60
84
95 ± 2
80 ± 2∗
7358 ± 396
5245 ± 255∗
0.97 ± 0.05
0.71 ± 0.03∗
133± 2
140 ± 3
2.66 ± 0.16
2.94 ± 0.15 847173111
High treadmill group subjects B, E, G and H, and low treadmill group subjects A, C, D, F and I; values are means ± S.E.M.;
n, no. of fibres studied. P0, peak isometric force; %, percentage of pre-flight value; V0, maximal shortening velocity; FL,
fibre length.∗Significantly different from pre-flight value, P < 0.05.
Table 6. Diameter, cross sectional area, peak force, and maximal shortening velocity of the gastrocnemius fast type II
fibre pre- and post-flight
DiameterCSA
Po
Vo
Condition
n
(μm)(%)(μm2) (%)(mN)(%)(kN m−2)(FL s−1)(%)
High treadmill
Pre-flight
Post-flight
Low treadmill
Pre-flight
Post-flight
All crew members
Pre-flight
Post-flight
52
58
91 ± 2
85 ± 2
9709 ± 361
5845 ± 221∗
0.97 ± 0.05
0.78 ± 0.03∗
147 ± 3
136 ± 3
3.45 ± 0.17
2.78 ± 0.13∗
93
87 8081
76
81
69 ± 2
70 ± 1
3970 ± 223
4012 ± 168
0.57 ± 0.03
0.61 ± 0.03
153 ± 4
154 ± 3
3.30 ± 0.14
2.76 ± 0.13∗
101101 107
84
128
139
78 ± 2
77 ± 1
5045 ± 282
4774 ± 154
0.73 ± 0.03
0.68 ± 0.02
151 ± 3
147 ± 2
3.36 ± 0.11
2.77 ± 0.09∗
999593
82
High treadmill group subjects B, E, G and H, and low treadmill group subjects A, C, D, F and I; values are means ± S.E.M.;
n, no. of fibres studied. P0, peak isometric force; %, percentage of pre-flight value; V0, maximal shortening velocity; FL,
fibre length.∗Significantly different from pre-flight value, P < 0.05.
4) or type II (Table6) fibres. The protective effect of the
treadmill countermeasure for the soleus type I fibre is
perhaps best appreciated by comparing the plot of fibre
atrophy versus force pre- and post-flight for each crew
member (Figs4 and 5). The low treadmill group showed
an average pre-flight force of ∼100mN and diameter of
100μm while post-flight the relationship for force versus
diameter was shifted down and to the left so that the
majorityofthefibresgeneratedapeakforce<100mNand
hadafibrediameter<100μm(Fig.4).Incomparison,the
down and left shift of the force versus diameter plot for
the high treadmill group was less apparent (Fig.5). The
average weekly treadmill running showed a significant
inverse correlation with thepercentage of soleus type I
fibre atrophy with r =0.68 (Fig.6). A second observation
that seemed to impact the extent of fibre atrophy and
hencethe decline in forcewas the pre-flightdiameter.The
larger the initial fibre diameter the greater the atrophy
such that a significant inverse correlation (r =0.87) was
observed between the mean pre-flight soleus type I fibre
diameter and the pre/post fibre diameter ratio expressed
as apercentage (Fig.6).
Figure7 plots the mean force (mN) versus diameter for
eachcrewmemberpre-(circles)andpost-flight(triangles)
for fast type II fibres from the soleus and gastrocnemius.
For the soleus, the plot was similar to that observed for
the slow fibre type as post-flight the force to diameter
relationship was shifted down (force loss) and to the left
(diameterloss)inthelowtreadmillgroupwithvirtuallyno
change in the plot (pre versus post) for the high treadmill
group. In contrast, the gastrocnemius fast type II fibres
were not protected by an increased amount of treadmill
running (Fig.7 and Table6).
Maximal shortening velocity (V0) and rate of tension
development (ktr)
With the exception of the soleus fast type II fibres, which
were unaltered by space flight (Table5), the maximal
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3576 R. H. Fitts and others
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-25
-20
-15
-10
-5
0
-60-50-40 -30-20-1010
0
Soleus Muscle Volume (% Change)
Soleus MHC I Fibre Atrophy (% change)
A
B
C
D
E
F
G
H
I
r=0.66
Figure 1. Relationship between soleus fibre atrophy and the
decline in whole muscle volume with prolonged space flight
The percentage change (pre- to post-flight) in the mean type I fibre
diameter is plotted versus the percentage change in soleus muscle
volume for each crew member. The crew members A–I are identified
by a specific colour with the low and high treadmill users indicated by
circles and squares, respectively. The variables showed a significant
(P < 0.05) correlation with an r = 0.66.
unloaded shortening velocity (V0) measured by the slack
test declined by ∼20% in both muscles and fibre types
studied (Tables3, 4 and 6). Unlike fibre size and force,
the decline in V0post-flight was not influenced by the
extent of treadmill running. The rate constants of tension
redevelopment (ktr) in slow type I fibres of the soleus and
gastrocnemiusmuscleswerenotsignificantlydifferentand
neither was altered by space flight (Table7).
Fibre stiffness
Peak fibre stiffness (E0), a property thought to reflect the
number of attached cross-bridges, increased in the slow
Figure 2. Representative pre- and post-flight fibre bundle
Cryostat cross sections of subject C pre-flight (A) and post-flight (B)
soleus muscle fibres were stained histochemically for actomyosin
ATPase activity after acid preincubation (method of Huckstorf et al.
2000). Slow fibres are darkly reactive, and fast fibres are lightly
reactive. Based on computerized digitizing planimetry, the post-flight
slow fibres are 31.5% smaller in cross-sectional area. Bar equals
75 μm for both panels.
fibre type in both the soleus and gastrocnemius muscles
(Table7). The peak force (P0)/peak stiffness (E0) ratio
was significantly less post-flight which suggests that if
the higher stiffness was caused by more cross-bridges, the
additional bridges were likely to be in a low force (weak
binding) state.
Vmax, peak power, and force and velocity
at peak power
Similar to V0(the maximal unloaded shortening velocity
measured by the slack test), the maximal shortening
velocity derived from the Hill plot of the force–velocity
relationship(Vmax)wassignificantlydepressedpost-flight
compared to pre-flight in the slow type I fibre from both
the soleus and the gastrocnemius (Tables8 and 9). Six
of the nine and five of nine crew members showed a
significant post-flight decline in type I fibre Vmaxin the
soleus and gastrocnemius muscles, respectively (Tables8
and 9).
Figure8 shows composite force–power relationships
for type I and II fibres of the soleus and gastrocnemius
muscles pre- and post-flight. Post-flight peak power of
the slow type I and fast type II fibre was significantly
depressedinboththesoleusandthegastrocnemius(Fig.8
and Tables8–10). As shown in Fig.8, the extent of the
decline in peak power of both fibre types was clearly
greaterinthesoleusthanthegastrocnemius.Interestingly,
while the fast type II fibres from both muscles showed
a significant post-flight drop in peak power (μNFLs−1),
when corrected for atrophy, the power expressed in watts
per litre was only depressed in the fibres isolated from the
gastrocnemius. The apparent explanation is that velocity
at peak power was significantly depressed only in gastro-
cnemius fibres (Table10).
In the case of the soleus but not the gastrocnemius
muscle, the high treadmill grouped showed less loss in
Figure 3. Relationship between microgravity induced fibre
atrophy and percentage increase in fast fibres in slow soleus
muscle
Symbols plot the percentage decrease in mean fibre diameter versus
the percentage increase in fast fibre type for each crew member. Each
subject A–I is colour coded as in Fig. 1.
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J Physiol 588.18
Prolonged space flight and human muscle 3577
Table 7. Peak stiffness (E0), P0/E0 ratio and the rate constant of tension
redevelopment (ktr) of the slow type I fibre pre- and post-spaceflight
Condition
E0
Po/E0
ktr
Sol Type I fibre
Pre-flight
Post-flight
GM Type I fibre
Pre-flight
Post-flight
2.16 ± 0.07 (146)
2.47 ± 0.09 (103)∗
62.6 ± 2.0
52.3 ± 1.9∗
1.58 ± 0.04 (164)
1.55 ± 0.04 (127)
2.22 ± 0.08 (90)
2.61 ± 0.12 (89)∗
64.3 ± 2.2
53.6 ± 2.4∗
1.47 ± 0.04 (111)
1.44 ± 0.03 (109)
Values are means ± S.E.M.; no. of fibres studied shown in parentheses; E0,
peak elastic modulus; P0, peak isometric force; ktr, rate constant of tension
redevelopment.∗Significantly different from pre-flight value, P < 0.05.
type I fibre peak power with a pre/post flight ratio of
63% compared to the low treadmill group mean of 48%.
Nine of nine and seven of eight crew members showed
a significant (P <0.05) post-flight decline in type I fibre
peak power (μNFLs−1) in the soleus and gastrocnemius,
respectively. When these data were expressed as Wl−1, the
post-flight peak power of the soleus and gastrocnemius
type I fibres remained depressed in five and three crew
Figure 4. Relationship between fibre diameter (μm) and peak Ca2+activated isometric force (mN) pre-
and post-flight for low treadmill group
Each symbol represents the result of a single soleus fibre. Type I fibres, blue diamonds. Type II fibres, red squares.
Hybrid Type I/II fibres, green triangles. Number of fibres for each fibre type and crew member are shown.
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3578 R. H. Fitts and others
J Physiol 588.18
members, respectively (Tables8 and 9). For the majority
of crew members, the decline in peak power expressed
as Wl−1was caused by a significant fall in both the force
(mN)andvelocity(FLs−1)elicitedatpeakpower(Tables8
and 9).
The two crew members with the greatest post-flight
loss in soleus muscle volume F (−23%) and A (−22%)
(see Trappe et al. 2009), also showed the greatest soleus
type I fibre atrophy (Table2), and loss of P0(Table3) and
absolute peak power (Table8). Crew member F showed a
post-flightincreaseinpeakpowerinWl−1indicatingthat
forthissubjectthelossofabsolutepeakpowerwasentirely
explained by the fibre atrophy and the accompanying loss
of force. Crew member A who displayed slightly less fibre
atrophy and loss of force than crew member F (Tables2
and 3), showed the greatest drop in soleus type I fibre
peak power (Table8). This can be explained in part by
a significant decline and increase in the a/P0 ratio for
crew members A and F, respectively (Table8). Due to the
greater curvature of the force–velocity relationship (i.e.
lower a/P0) post-flight, the mean force as a percentage of
P0at peak power for crew member A’s fibres was lower
(14%ofP0)thanthepre-flightcondition(16%ofP0)and
this contributed to the reduced power. The opposite was
trueofcrewmemberFasthea/P0ratioforthisindividual’s
soleus type I fibres was significantly elevated post-flight
Figure 5. Relationship between fibre diameter (μm) and peak Ca2+activated isometric force (mN) pre-
and post-flight for high treadmill group
Each symbol represents the result of a single soleus fibre. Type I fibres, blue diamonds. Type II fibres, red squares.
Hybrid Type I/II fibres, green triangles. Number of fibres for each fibre type and crew member are shown.
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J Physiol 588.18
Prolonged space flight and human muscle3579
Figure 6. Correlation of soleus type I fibre atrophy with
treadmill running and pre-flight fibre diameter
A, relationship between microgravity-induced fibre atrophy and
amount of treadmill running (min week−1). Symbols plot the
percentage decrease in mean fibre diameter versus amount of
treadmill running (min week−1) for each crew member. B, relationship
between pre-flight fibre diameter (μm) and percentage fibre atrophy.
Symbols plot the mean pre-flight fibre diameter versus percentage
fibre atrophy. For both the top and bottom plots, each subject is
colour coded as shown in Fig. 1. The circles and squares indicate low
and high treadmill groups, respectively.
(Table8).Inaddition,crewmemberAshowedasignificant
dropinvelocityatpeakpower,whilecrewmemberFdidn’t
(Table8).
Force–pCa relationship
The force–pCa relationship in the slow type I and fast
type II fibres pre- and post-flight are shown in Table11.
In the slow but not the fast fibre type, the slope of the
Hill plot for values less than half-maximal activation (n2)
were significantly higher post-flight, and this was true for
boththesoleusandgastrocnemiusmuscles(Table11).The
pCa50for the slow type I fibre in both muscles showed a
small but significant increase (Table11). The post-flight
increase in the n2of the type I fibre was not caused by the
expression of the fast troponin or tropomyosin isoforms
as SDS gel analysis of the post-flight fibres showed only
thecardiac/slowfibreformoftroponinC(cTn-C)andthe
slow isoform of Tn-I, Tn-T and tropomyosin (Fig.9).
Ultrastructural changes and quantification
At the electron microscopic level, crew members
exhibiting high levels of slow fibre atrophy in the soleus
muscle following 6months of spaceflight had smaller
myofibrils, fewer intracellular lipids, smaller and globular
mitochondria and increased space between myofibrils
filledwithglycogenparticles(Fig.10).Conversely,subjects
Figure 7. Relationship between fibre diameter (μm) and peak Ca2+activated isometric force (mN) pre-
and post-flight for type II fibres
Each subject is colour coded as shown in Fig. 1. The average pre- and post-flight values are represented by circles
and triangles, respectively. The plots show soleus type II fibres for the low (top left) and high (top right) treadmill
groups, and gastrocnemius type II fibres from the low (bottom left) and high (bottom right) treadmill groups.
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3580 R. H. Fitts and others
J Physiol 588.18
Table 8. Vmax, peak power, force and velocity at peak power for soleus slow type I fibre pre- and post-flight
Crew Member
Vmax
a/P0
Peak power Force at PPVel at PP
n
(FL s−1)(μN FL s−1) (%)(W l−1) (mN)(FL s−1)
A
Pre-flight
Post-flight
49
25
0.91 ± 0.05
0.77 ± 0.05∗
0.036 ± 0.002
0.028 ± 0.002∗
19.39 ± 1.39
5.36 ± 0.29∗
2.04 ± 0.09
2.04 ± 0.08
0.137 ± 0.006
0.052 ± 0.002∗
0.139 ± 0.006
0.103 ± 0.005∗
28
B
Pre-flight
Post-flight
19
11
0.88 ± 0.05
0.76 ± 0.04
0.038 ± 0.002
0.033 ± 0.002
10.37 ± 0.75
7.35 ± 0.39∗
2.64 ± 0.13
1.60 ± 0.07∗
0.075 ± 0.005
0.064 ± 0.003
0.136 ± 0.004
0.114 ± 0.005∗
71
C
Pre-flight
Post-flight
25
28
0.95 ± 0.06
0.93 ± 0.07
0.029 ± 0.001
0.033 ± 0.003
20.86 ± 2.61
11.13 ± 0.84∗
2.31 ± 0.20
2.11 ± 0.16
0.146 ± 0.009
0.084 ± 0.003∗
0.134 ± 0.008
0.129 ± 0.007 53
D
Pre-flight
Post-flight
32
28
1.04 ± 0.05
0.83 ± 0.05∗
0.024 ± 0.001
0.030 ± 0.002∗
17.95 ± 1.10
9.45 ± 0.68∗
2.26 ± 0.11
1.90 ± 0.09∗
0.132 ± 0.004
0.079 ± 0.003∗
0.133 ± 0.006
0.117 ± 0.006∗
53
E
Pre-flight
Post-flight
26
40
0.90 ± 0.07
0.71 ± 0.03∗
0.026 ± 0.002
0.031 ± 0.002
13.08 ± 1.19
9.45 ± 0.38∗
1.43 ± 0.05
1.59 ± 0.06
0.110 ± 0.007
0.093 ± 0.003∗
0.117 ± 0.007
0.101 ± 0.003∗
72
F
Pre-flight
Post-flight
29
6
0.87 ± 0.06
0.61 ± 0.09
0.025 ± 0.001
0.051 ± 0.010∗
17.11 ± 0.83
7.00 ± 1.83∗
1.50 ± 0.08
2.70 ± 0.67∗
0.157 ± 0.007
0.061 ± 0.006∗
0.113 ± 0.007
0.110 ± 0.00441
G
Pre-flight
Post-flight
38
26
0.78 ± 0.04
0.56 ± 0.03∗
0.024 ± 0.001
0.032 ± 0.002∗
9.94 ± 0.64
5.90 ± 0.50∗
1.35 ± 0.08
1.05 ± 0.05∗
0.103 ± 0.005
0.079 ± 0.008∗
0.100 ± 0.005
0.080 ± 0.003∗
59
H
Pre-flight
Post-flight
45
38
0.90 ± 0.05
0.52 ± 0.03∗
0.025 ± 0.001
0.033 ± 0.002∗
9.09 ± 0.34
4.40 ± 0.28∗
1.55 ± 0.06
0.98 ± 0.07∗
0.082 ± 0.004
0.058 ± 0.003∗
0.117 ± 0.005
0.077 ± 0.004∗
48
I
Pre-flight
Post-flight
High Treadmill (4)
Pre-flight
Post-flight
Low Treadmill (5)
Pre-flight
Post-flight
All Crew
Pre-flight
Post-flight
48
37
0.72 ± 0.03
0.46 ± 0.02∗
0.024 ± 0.001
0.032 ± 0.001∗
10.02 ± 0.45
6.48 ± 0.48∗
1.34 ± 0.07
1.00 ± 0.06∗
0.111 ± 0.005
0.094 ± 0.005∗
0.091 ± 0.003
0.067 ± 0.003∗
65
128
114
0.86 ± 0.03
0.62 ± 0.02∗
0.027 ± 0.001
0.032 ± 0.001∗
10.35 ± 0.57
6.77 ± 0.28∗
1.63 ± 0.05
1.26 ± 0.04∗
0.093 ± 0.003
0.076 ± 0.003∗
0.115 ± 0.003
0.089 ± 0.002∗
63 ± 6
183
124
0.88 ± 0.02
0.76 ± 0.04∗
0.028 ± 0.001
0.031 ± 0.001
16.52 ± 0.64
7.98 ± 0.35∗
1.84 ± 0.05
1.77 ± 0.07
0.134 ± 0.003
0.077 ± 0.002∗
0.121 ± 0.003
0.104 ± 0.004∗
48 ± 6
311
238
0.88 ± 0.02
0.68 ± 0.02∗
0.027 ± 0.001
0.032 ± 0.001∗
14.20 ± 0.45
7.47 ± 0.24∗
1.76 ± 0.04
1.53 ± 0.05∗
0.117 ± 0.002
0.077 ± 0.002∗
0.119 ± 0.002
0.097 ± 0.002∗
54 ± 5
High treadmill group subjects B, E, G and H, and low treadmill group subjects A, C, D, F and I; values are means ± S.E.M.; n, no. of fibres
studied. Vmax, maximal unloaded shortening velocity determined from the Hill plot; a/Po, unitless parameter describing curvature of
the force–velocity relationship. The relative power unit of watts per litre (W l−1) is equivalent of kN m−2FL s−1; Vel at PP, velocity at
peak power; Force at PP, force at peak power; FL, fibre length; no. of high and low treadmill crew members listed in parentheses.
∗Significantly different from pre-flight value, P < 0.05.
with little atrophy retained similar sized, closely packed
myofibrils, lipid droplets and filamentous mitochondria
encircling the myofibrils at the I band level (Fig.10).
Within the myofibrils in the A bands after spaceflight,
individual thick filaments in the near M band region
were surrounded by a greater number of thin filaments
compared to pre-flight (Fig.11). The average thin
filament density (2744±167μm−2) post-flight in the
near M region was 22% (P <0.01) higher than that
(2253±132μm−2) before flight. Two subjects did not
show high thin filament density post-flight (Fig.12). The
ratio of thin:thick filaments was significantly (P <0.01)
higher post-flight (2.9±0.21) compared to pre-flight
(2.2±0.15).Theincreaseinthinfilamentsandamountof
fibre atrophy were directly correlated, although the rise in
filament density plateaued around 3200filamentsμm−2,
indicating an upper limit (Fig.13). This limit value
matches our previously published data (Riley et al. 1998,
2000)forthinfilamentdensitiesinsoleusIbandsnearthe
Zline,whichaveraged3251μm−2forthetwostudies(the
published density numbers normalized to 2.4μm were
renormalized to 2.5μm sarcomere length for comparison
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Page 15

J Physiol 588.18
Prolonged space flight and human muscle 3581
Table 9. Vmax, peak power, force and velocity at peak power for gastrocnemius slow type I fibre pre- and post-flight
Crew member
Vmax
a/P0
Peak power Force at PPVel at PP
n
(FL s−1)(μN FL s−1) (%) (W l−1) (mN)(FL s−1)
A
Pre-flight
Post-flight
42
26
0.87 ± 0.04
0.72 ± 0.06∗
0.036 ± 0.002
0.034 ± 0.003
10.04 ± 0.59
5.64 ± 0.34∗
2.34 ± 0.10
2.15 ± 0.10
0.074 ± 0.003
0.054 ± 0.002∗
0.133 ± 0.005
0.103 ± 0.006∗
56
C
Pre-flight
Post-flight
10
10
0.64 ± 0.03
0.64 ± 0.04
0.038 ± 0.003
0.032 ± 0.003
12.07 ± 0.86
6.90 ± 0.44∗
1.56 ± 0.09
1.25 ± 0.06∗
0.120 ± 0.009
0.074 ± 0.006∗
0.100 ± 0.003
0.093 ± 0.00457
D
Pre-flight
Post-flight
28
24
0.86 ± 0.06
0.78 ± 0.10
0.027 ± 0.001
0.030 ± 0.002
13.48 ± 0.85
7.24 ± 0.44∗
1.79 ± 0.09
1.71 ± 0.09
0.115 ± 0.004
0.071 ± 0.004∗
0.116 ± 0.005
0.105 ± 0.00854
E
Pre-flight
Post-flight
24
26
1.08 ± 0.05
0.69 ± 0.05∗
0.024 ± 0.001
0.032 ± 0.002∗
16.21 ± 0.86
8.85 ± 0.39∗
1.88 ± 0.13
1.47 ± 0.06∗
0.117 ± 0.006
0.091 ± 0.004∗
0.139 ± 0.005
0.099 ± 0.005∗
55
F
Pre-flight
Post-flight
40
12
0.75 ± 0.04
0.69 ± 0.07
0.025 ± 0.001
0.030 ± 0.003
7.37 ± 0.41
5.70 ± 0.23∗
1.28 ± 0.05
1.50 ± 0.07∗
0.079 ± 0.004
0.061 ± 0.003∗
0.097 ± 0.005
0.094 ± 0.00677
G
Pre-flight
Post-flight
33
25
0.79 ± 0.04
0.51 ± 0.04∗
0.022 ± 0.001
0.036 ± 0.005∗
6.30 ± 0.28
3.40 ± 0.27∗
1.32 ± 0.06
1.01 ± 0.09∗
0.064 ± 0.002
0.050 ± 0.005∗
0.100 ± 0.005
0.074 ± 0.004∗
54
H
Pre-flight
Post-flight
32
11
0.77 ± 0.04
0.60 ± 0.07∗
0.027 ± 0.002
0.037 ± 0.005∗
7.31 ± 0.36
4.94 ± 0.44∗
1.42 ± 0.07
1.41 ± 0.16
0.073 ± 0.003
0.058 ± 0.006∗
0.101 ± 0.004
0.087 ± 0.005∗
68
I
Pre-flight
Post-flight
High treadmill (3)
Pre-flight
Post-flight
Low treadmill (5)
Pre-flight
Post-flight
All crew
Pre-flight
Post-flight
29
24
0.82 ± 0.04
0.52 ± 0.04∗
0.025 ± 0.001
0.034 ± 0.003∗
4.69 ± 0.22
4.11 ± 0.24
1.45 ± 0.11
1.31 ± 0.08
0.045 ± 0.002
0.056 ± 0.003∗
0.106 ± 0.004
0.073 ± 0.004∗
88
89
62
0.86 ± 0.03
0.60 ± 0.03∗
0.024 ± 0.001
0.034 ± 0.002∗
9.34 ± 0.53
5.96 ± 0.38∗
1.51 ± 0.05
1.27 ± 0.06∗
0.082 ± 0.003
0.069 ± 0.004∗
0.111 ± 0.003
0.087 ± 0.003∗
59 ± 5
149
96
0.81 ± 0.02
0.67 ± 0.03∗
0.029 ± 0.001
0.032 ± 0.001
9.07 ± 0.36
5.80 ± 0.20∗
1.73 ± 0.05
1.66 ± 0.05
0.081 ± 0.003
0.062 ± 0.002∗
0.113 ± 0.003
0.094 ± 0.003∗
66 ± 7
238
158
0.83 ± 0.02
0.64 ± 0.02∗
0.027 ± 0.001
0.033 ± 0.001∗
9.17 ± 0.30
5.86 ± 0.19∗
1.64 ± 0.04
1.51 ± 0.04
0.081 ± 0.002
0.065 ± 0.002∗
0.112 ± 0.002
0.091 ± 0.002∗
64 ± 5
High treadmill group subjects B, E, G and H, and low treadmill group subjects A, C, D, F and I; values are means ± S.E.M.; n, no. of fibres
studied. Vmax, maximal unloaded shortening velocity determined from the Hill plot; a/Po, unitless parameter describing curvature of
the force–velocity relationship. The relative power unit of watts per litre (W l−1) is equivalent of kN m−2FL s−1; Vel at PP, velocity
at peak power; Force at PP, force at peak power; FL, fibre length; no. of high and low treadmill crew members listed in parentheses.
∗Significantly different from pre-flight value, P < 0.05.
with current data). The 3251μm−2represents the largest
number nucleated at the Z band (Riley et al. 1998,
2000). For the 180-day pre- and post-flight slow fibres,
thin filament density and shortening V0were inversely
related (Fig.14A). In contrast, the mean actin/myosin
ratio determined from SDS band densities was unaltered
pre- (1.26±0.05) to post-flight (1.29±0.07), and the
correlation between the actin/myosin ratios and thin
filament densities was not significant (Fig.14B). Of the
five crew members showing an increased thin filament
density, three showed a small increase and two a small
decrease in the actin/myosin ratio post-flight (Fig.14B).
Therelationshipofthinfilamentdensityandtreadmilluse
suggested that high treadmill use was partially effective in
preventingincreasedthinfilamentdensity(Fig.15A).The
level of cycle use also prevented in part the increase in
post-flight thin filament density (Fig.15B).
Discussion
FrompreviousMIR missionsand our recent observations
on International Space Station crew members, it is well
established that significant losses in leg muscle mass
occur with prolonged space flight, and that considerable
variability in the extent of muscle atrophy exists between
individuals(Fittsetal.2000;Trappeetal.2009).Theresults
of this study present the first cellular analysis of the effects
oflongdurationspaceflightonthestructureandfunction
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