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

The Journal of PhysiologyVolume 588, Issue 18 p. 3567-3592 Free Access Prolonged space flight-induced alterations in the structure and function of human skeletal muscle fibres R. H. Fitts, R. H. Fitts Marquette University, Milwaukee, WI 53201-1881, USASearch for more papers by this authorS. W. Trappe, S. W. Trappe Ball State University, Muncie, IN 47306, USASearch for more papers by this authorD. L. Costill, D. L. Costill Ball State University, Muncie, IN 47306, USASearch for more papers by this authorP. M. Gallagher, P. M. Gallagher Ball State University, Muncie, IN 47306, USASearch for more papers by this authorA. C. Creer, A. C. Creer Ball State University, Muncie, IN 47306, USASearch for more papers by this authorP. A. Colloton, P. A. Colloton Marquette University, Milwaukee, WI 53201-1881, USASearch for more papers by this authorJ. R. Peters, J. R. Peters Marquette University, Milwaukee, WI 53201-1881, USASearch for more papers by this authorJ. G. Romatowski, J. G. Romatowski Marquette University, Milwaukee, WI 53201-1881, USASearch for more papers by this authorJ. L. Bain, J. L. Bain Medical College of Wisconsin, Milwaukee, WI 53226, USASearch for more papers by this authorD. A. Riley, D. A. Riley Medical College of Wisconsin, Milwaukee, WI 53226, USASearch for more papers by this author R. H. Fitts, R. H. Fitts Marquette University, Milwaukee, WI 53201-1881, USASearch for more papers by this authorS. W. Trappe, S. W. Trappe Ball State University, Muncie, IN 47306, USASearch for more papers by this authorD. L. Costill, D. L. Costill Ball State University, Muncie, IN 47306, USASearch for more papers by this authorP. M. Gallagher, P. M. Gallagher Ball State University, Muncie, IN 47306, USASearch for more papers by this authorA. C. Creer, A. C. Creer Ball State University, Muncie, IN 47306, USASearch for more papers by this authorP. A. Colloton, P. A. Colloton Marquette University, Milwaukee, WI 53201-1881, USASearch for more papers by this authorJ. R. Peters, J. R. Peters Marquette University, Milwaukee, WI 53201-1881, USASearch for more papers by this authorJ. G. Romatowski, J. G. Romatowski Marquette University, Milwaukee, WI 53201-1881, USASearch for more papers by this authorJ. L. Bain, J. L. Bain Medical College of Wisconsin, Milwaukee, WI 53226, USASearch for more papers by this authorD. A. Riley, D. A. Riley Medical College of Wisconsin, Milwaukee, WI 53226, USASearch for more papers by this author First published: 16 September 2010 https://doi.org/10.1113/jphysiol.2010.188508Citations: 218 Corresponding author R. H. Fitts: Marquette University, Department of Biological Sciences, PO Box 1881, Milwaukee, WI 53201-1881, USA. Email: [email protected] AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL 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. Abbreviations CSA cross-sectional area FL fibre length ISS International Space Station k tr rate constant of tension redevelopment P 0 peak force V 0 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 from the last 40 years of space research, particularly studies 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 muscle is particularly susceptible to microgravity-induced deterioration in both structure and function (Convertino, 1990; Fitts et al. 2000; Fitts et al. 2001). A consistent observation is significant atrophy of both upper and lower leg muscles with the response occurring more rapidly in the triceps surae muscle group (ankle plantar flexors) 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 post-flight values. The authors concluded that space flight 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 15 and 26% smaller post- compared to pre-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 microgravity steady state may be reached by approximately 180 days (Fitts et al. 2000). The loss in muscle force primarily reflects the decline in mass. Consequently, when single fibre force is expressed relative to cross-sectional area, there is little difference between pre- and post-flight values (Widrick et al. 1999; Fitts et al. 2000). In addition to 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; Fitts et al. 2000). For example, after 31 days in space, 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 V0 was associated with and likely caused by an increase in myofilament lattice spacing that resulted from a reduction in thin filament density. It is unknown whether or not this adaptation persists with prolonged space flight or reflects a transient response to short duration flight. A hallmark of space flight is that considerable variability 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 the calf from 7 to 20% (Trappe et al. 2009). Similarly, Zange et al. (1997) observed muscle mass losses with 6 months 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+ required for half-maximal activation of soleus type I fibres post-flight ranged from no difference to 0.31 μmol 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 (∼180 days) 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 (Increment 5) originated and landed in Russia aboard 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 were 45 ± 2 years, 176 ± 2 cm, 81 ± 3 kg, and 177 ± 4 days (range = 161–192 days), 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 accordance with the Human Subjects Institutional Review 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 (Interim Resistive Exercise Device; iRED). The treadmill could be used in a passive (subject driven) or active (motorized) mode of operation. The exercise countermeasure 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 Table 1. 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 by NASA and the Russian Space Agencies (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). 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) Exercises Frequency Sets/reps 138 ± 26 126 ± 10 146 ± 32 3.2 ± 0.5 Squats 3–6 days week−1 12–20 Range: Range: Range: Range: Heel raises 3–6 days week−1 12–20 Little – 296 102 – 150 64 – 312 2.1 – 5.5 Dead lifts 3–6 days week−1 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). Muscle biopsy A muscle biopsy (Bergstrom, 1962) of ∼80 mg 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 approximately 6–8 h after landing. The post-flight activity 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 subsequent 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 (125 mm potassium propionate, 20 mm imidazole, 2 mm EGTA, 4 mm ATP, 1 mm 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, water ice bottles in two boxes with each containing two vials (one soleus and one gastrocnemius sample), hand carried back to Ball State and Marquette Universities. Upon arrival, the bundles were placed in fresh skinning solution and stored at −20°C for up to 4 weeks. All contractile measurements on fibres from a given muscle were performed within 4 weeks of the initial bundle isolation. A third portion of each biopsy was pinned at a mild stretch and immersion fixed in a 0.1 m cacodylate buffer (pH 7.2) containing 4% glutaraldehyde and 2% paraformaldehyde with 5 mm 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 constants adjusted 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 180 mm, 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. 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 bundle was removed from the skinning solution and transferred to a dissection chamber containing pH 7.0 relaxing solution (4°C). An individual fibre was gently isolated from the bundle, transferred to an ∼1 ml glass-bottomed chamber milled in a stainless steel plate. While submerged 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 Instruments data acquisition board (NI-DAQ). To disrupt any remaining intact membranes, the fibre was submerged into a relaxing solution containing 0.5% Brij 58 for 30 s after which the fibre bath was exchanged twice with relaxing solution. The experimental chamber was mounted on the stage of an inverted microscope. Sarcomere length was adjusted to 2.5 μm using an eyepiece micrometer (800×), and the length of the fibre (FL) was recorded. A digital photo (Pro IDEO CVC-140 camera) was taken of the fibre while it was briefly suspended in air. Fibre diameter was determined at three points along the length of the fibre using Scion Image software, and fibre CSA calculated from the mean diameter 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 (determined from the y-axis intercept of slack test) exceeded 10% (Trappe et al. 2004). For most fibres, P0 declined <10% from the beginning to the end of the experiment. Contractile function of individual fast type II 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 fibre length. Each fibre was subjected to five different slack steps and fibre V0 (FL s−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) used a laser to clamp the sarcomeres at 2.5 μm. The clamp procedure is time consuming and not practical when hundreds of fibres are studied. In preliminary studies, we determined that the ktr of the slow type I fibre was identical with and without a laser clamp, while the fast type II fibre showed a significantly lower ktr in the absence of a laser clamp (Fitzsimons et al. 2001). Thus, we determined the ktr in slow but not fast fibres. The measurement requires activation of the fibre in pCa 4.5 and following attainment of steady tension a 400 μm slack, a 40 ms 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 rate constant k is ktr and thought to reflect the rate limiting step in the generation of the high force state (Metzger & Moss, 1990). Following the determination of V0 and ktr, isotonic load 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-fitting procedure (Marquardt–Levenberg 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 P0 in increments of 1%. In a subset of fibres (see Table 11 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) pCa50 determined (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= (Δforce in activating solution −Δforce in relaxing solution/Δlength) (fibre length/fibre cross-sectional area). Table 11. Force–pCa relationship in slow type I and fast type II fibres pre- and post-spaceflight Variable SOL Type I GM Type I Type II Pre-flight Post-flight Pre-flight Post-flight Pre-flight Post-flight n 125 98 76 80 31 49 Activation threshold 6.96 ± 0.02 6.94 ± 0.03 6.85 ± 0.03 6.90 ± 0.03 6.63 ± 0.05 6.56 ± 0.03 pCa50 5.94 ± 0.02 6.00 ± 0.02* 5.86 ± 0.02 5.97 ± 0.03* 6.10 ± 0.04 6.09 ± 0.03 n 1 1.60 ± 0.05 1.72 ± 0.06 1.73 ± 0.07 1.89 ± 0.09 1.76 ± 0.20 2.14 ± 0.15 n 2 2.35 ± 0.04 2.72 ± 0.07* 2.43 ± 0.06 2.79 ± 0.09* 5.04 ± 0.38 5.49 ± 0.34 Values are means ±s.e.m.; n, no. of fibres studied; all values are pCa or –log of the Ca2+ concentration and pCa50, –log of the Ca2+ concentration at half-maximal activation; n1 and n2, slope of Hill plot for values greater than and less than half-maximal activation, respectively. The type II data are composite means for all fast fibres from soleus and gastrocnemius muscles. *Significantly different from pre-flight value, P < 0.05. SDS gel analysis of actin and myosin composition After the contractile tests, the fibre was removed from the experimental set-up and solubilized in 10 μl of sodium dodecyl sulfate (SDS) sample buffer (6 mg ml−1 EDTA, 0.06 m tris(hydroxymethyl)aminomethane, 1% SDS, 2 mg ml−1 Bromophenol Blue, 15% glycerol, 5%β-mercaptoethanol), and stored at −80°C. Fibre types were identified by the myosin heavy chain (MHC) isoform pattern using 5% polyacrylamide gels as slow type I or fast type II. Fibres containing both slow and fast myosin (hybrid fibres) were not included in the analysis. Myosin (heavy and light chains), actin, tropomyosin and troponin profiles were determined by 12% 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/myosin ratio was determined on an average of 26 ± 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. Figure 14Open in figure viewerPowerPoint Correlations of fibre V0 and actin content with thin filament density In pre- and post-flight soleus muscles, the shortening velocity (V0) of type I fibres is low when thin filament density is high and the correlation is significant at P < 0.05 (A), while the actin/myosin ratio of type I fibres did not change pre- to post-flight and the correlation with thin filament density was not significant (B). Each subject is colour coded as shown in Fig. 1 with the filled symbols pre-flight and the partially filled post-flight. 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 filament density was highest in the I band and fell off in the A band because the short filaments arising from the Z line were not long enough to reach the A band in a sarcomere at 2.5 μm length. For the present 180-day spaceflight muscles, a qualitative inspection of thin filament numbers in the I band near the Z line and within the A band (overlap A) where thin filaments first overlap thick filaments indicated that thin filaments were not missing as expected from 17 day flight data but appeared more abundant, pointing to increased thin filament length. Further into the A band nearer the M line, thin filament number decreased, which made sense because only the longest thin filaments (∼1.27 nm) could reach this far. Thus, to detect increased thin filament density due to increased thin filament length, 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. The concepts of thin filament length, thin filament density and location of near the M line measurement site are illustrated diagrammatically in Fig. 16. Figure 16Open in figure viewerPowerPoint Conceptual diagram of increasing thin filament density following prolonged spaceflight The sarcomere at 2.5 μm illustrates thin filament (blue line) density and lengths in a pre-flight muscle (left half) and post-flight density and lengths in the right half. Thin filaments arise from nucleation sites in the Z band and exhibit different lengths in normal skeletal muscles. The density of thin filaments is highest near the Z band (6 filaments in the Near Z region) and progressively decreases away from the Z band because some filaments are too short to overlap the A band (4 in Overlap A) and others end before reaching the Near M band region. After prolonged spaceflight, thin filament length increases (indicated by red extensions of thin filaments) and density increases in the Near M region (post-flight). Cross sections (70 nm) 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 31.3 nm (2.5 μm sarcomere length). After normalization, the average thick filament density of the pre-flight fibres (999 ± 21 filaments μm−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 0.0056 μm2 grid square at ×201,000 magnification on the computer screen using Gunderson's rules for sampling (Riley et al. 2000, 2005). For non-biased sampling of thick and thin filament counts, the grid squares were positioned at random over the A band regions of thick and thin filament overlap within an estimated 100–300 nm of the M band in central myofibrils (Riley et al. 2002). The position of the sampling