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Human Performance Laboratory, Ball State University, Muncie, Indiana
Submitted 23 May 2007 ; accepted in final form 16 July 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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skeletal muscle; contractile properties; microgravity; spaceflight; WISE-2005 study
500 bed rest subjects in the last 40 years have been women, with no studies focusing exclusively on women. Because women will certainly be involved in future spaceflight missions, more information is needed in this population to better understand how skeletal muscle responds to prolonged unloading and exercise. Furthermore, information on muscle atrophy in women would be applicable to the general health care industry for various clinical conditions (i.e., cachexia, sarcopenia). Developing specific countermeasures to negate the unloading-induced atrophy at the whole muscle and cellular levels is warranted to promote long-term space exploration and for its potential use in clinical situations. A recent 90-day bed rest study involving male volunteers tested the efficacy of a resistance-training countermeasure to combat reduced skeletal muscle function and mass that occur during unloading (3). Although whole muscle strength and mass were maintained by resistance exercise during bed rest, cellular data from the vastus lateralis of resistance-trained subjects showed an incomplete preservation of slow-twitch [myosin heavy chain (MHC) I] muscle fiber size and function (46). In addition, a slow-to-fast shift in MHC isoform composition with a concomitant increase in fibers expressing multiple MHC isoforms (i.e., hybrid fibers) was also observed (21). These findings highlight that resistance exercise alone was insufficient to maintain slow-twitch fiber structure and function during long-duration bed rest in men. As a result, a more comprehensive exercise countermeasures program needs to be identified that effectively targets and protects both slow- and fast-twitch muscle fibers in human skeletal muscle. This information will be important to ensure the safety and work capacity of humans engaged in long-duration space travel and planetary exploration.
One possible solution would be the integration of aerobic exercise into the resistance training program, since aerobic training mainly targets slow-twitch muscle fibers (41) and has historically been part of the on-orbit exercise countermeasure program. Studies investigating the effects of concurrent resistance and endurance exercise have reported an inhibitory effect on strength and hypertrophy compared with resistance training alone (4, 15, 27). Although potential interference between resistance and endurance training may prevent optimal muscle strength and mass gains, a concurrent training program does improve whole muscle strength and size (4, 15, 27). At the single fiber level, concurrent training has been shown to preserve or increase slow- and fast-twitch muscle fiber size (4, 25, 38), as well as decrease hybrid muscle fiber types (38). To the best of our knowledge, concurrent training protocols as a countermeasure for skeletal muscle during prolonged unloading have not been investigated. The findings derived from ground-based training studies suggest that the combination of a resistance and aerobic exercise training regimen may provide the balanced stimulus necessary to fully protect unloaded human skeletal muscle by targeting both MHC I and IIa fibers and attenuating a slow-to-fast shift in MHC isoform composition.
The anabolic effect of amino acids (39, 51) has recently received attention as a potential countermeasure for skeletal muscle during periods of unloading and would be advantageous in situations where exercise may not be possible. Previous studies have shown that amino acids stimulate muscle protein synthesis (8, 9, 12, 34). Independently, the amino acid leucine has been shown to have strong anabolic (10, 29, 30, 40) and anti-catabolic (10, 13, 20, 36, 44) effects on skeletal muscle protein kinetics. Shorter duration bed rest studies support the idea for a nutritional countermeasure to promote the preservation of muscle mass during unloading (7, 37, 42, 43). In a recent 28-day bed rest study, a nutritional countermeasure consisting of additional amino acids resulted in conflicting results showing that whole body lean muscle mass was maintained (37) but not vastus lateralis MHC I and IIa fiber size (19). To date, the efficacy of an amino acid countermeasure has not been tested in longer duration bed rest or in women during periods of unloading.
The primary goal of this investigation was to test the hypothesis that a concurrent training protocol consisting of resistance and aerobic exercise would preserve vastus lateralis MHC I and IIa single muscle fiber structure and contractile function. A secondary objective of this investigation was to test the hypothesis that a leucine-enriched high-protein diet would partially preserve vastus lateralis MHC I and IIa muscle fiber size and contractile function. A more complete report of the whole muscle performance and mass are presented by our research team elsewhere (48).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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This investigation integrated several physiological, metabolic, and psychological experiments to examine the responses to prolonged bed rest (i.e., simulated weightlessness) in women. The primary goal of this study was to test specific exercise and nutritional countermeasure programs to the expected deleterious effects of long-term bed rest. The study was conducted at the Institute for Space Physiology and Medicine (MEDES) in Toulouse, France.
The study design consisted of 20 days of baseline data collection (BDC), 60 days of 6° head-down tilt (HDT) bed rest, and a recovery period that varied in duration depending on the specific measurements. The BDC period provided time for the subjects to equilibrate to the standardized diet during BDC for each of the volunteers. All meals and snacks were prepared for the volunteers. Body weight was measured daily (during bed rest in the 6° HDT position). During bed rest, the volunteers remained in the 6° HDT position or horizontal position for all activities, including eating, bathing, excretory function, and physiological testing and training (0° for treadmill). Continuous video and medical staff monitoring ensured compliance to the bed rest period.
The study was completed in two separate campaigns, with each campaign consisting of 12 subjects, four from each experimental group: bed rest only (BR), bed rest with the exercise countermeasure (BRE), and bed rest with the nutritional countermeasure (BRN). The BDC, bed rest, and initial recovery periods of both campaigns were completed in one calendar year (2005). Because similar changes were observed in campaign 1 and campaign 2, the data were combined for each group.
Subjects
Twenty-four healthy women from the European Union were recruited and underwent 60 days of 6° HDT bed rest. Subjects were divided into bed rest only (n = 8), bed rest + nutritional countermeasure (n = 8), or bed rest + exercise countermeasure (n = 8) groups. Subject characteristics are shown in Table 1. Screening for potential volunteers took place at the MEDES clinic and involved an interview, general medical examination, and psychological evaluation. Subjects were selected into each group by the MEDES staff based on their medical and psychological profiles. Subjects were recreationally active (pre-bed rest maximal oxygen consumption of 39 ± 1 ml·kg–1·min–1 for all groups) and excluded from participation if they were sedentary or too involved with physical training. Before the screening, each potential volunteer was informed of all procedures and potential risks associated with the experimental testing. Informed consent was then obtained from each volunteer. This study was approved by the Human Use Committees in France and the United States (Johnson Space Center and Ball State University).
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Resistance training protocol. The BRE group trained the thigh muscle groups using supine squat (SS) exercises on an inertial ergometer (2, 3, 5). Resistance exercise was scheduled for each subject approximately every third day (2–3 days/wk) beginning on day 2 of bed rest for a total of 19 sessions. The inertial ergometer was in the 6° HDT position, and all resistance exercises were performed in the supine position. Ten minutes of light supine cycling and submaximal SS repetitions were completed as warm-up. The SS exercise consisted of four sets of seven maximal concentric and eccentric repetitions. There were 2 min of rest between sets. Force and flywheel rotational velocity were measured with work and power calculated throughout each repetition (2, 3, 5). This exercise protocol was similar to a previous 90-day study conducted in men (3, 46).
Because of various medical aspects that arose during the bed rest period, including the combined aerobic and resistance exercise countermeasure program (soreness and injury) and occasional mild illness, not all sessions were completed as planned. To help minimize the chance for injury, the second bed rest campaign (half the subjects) had a ramp-up phase for the resistance exercise sessions. The first three sessions were at 70, 80, and 90% of maximal effort, with all remaining sessions planned as maximal effort. Data compiled at the end of both bed rest campaigns provided a profile of the exercise sessions. For the first campaign, 76% were conducted as planned, 16% were reduced effort, and 8% were missed. For the second campaign, 88% were conducted as planned, 9% were reduced effort, and 3% were missed. The submaximal and missed resistance exercise sessions varied among the volunteers and were scattered throughout the 60-day bed rest period.
Aerobic training protocol. The lower body negative pressure (LBNP) treadmill device used for this study was similar to that described previously (11, 32, 49). Two to four days per week, exercise subjects performed 40 min of exercise ranging from 40% to 80% of pre-bed rest peak oxygen consumption, followed by 10 min of resting LBNP (32, 49).
During the course of the 60-day bed rest, 29 exercise sessions were prescribed for each BRE subject. Not all exercise sessions were completed by all subjects due to illness, joint pain, and soreness from prior exercise. One subject was unable to complete the first two exercise sessions due to medical reasons but subsequently completed all other sessions. In one subject, three exercise sessions were not completed because of back or hip pain. One exercise session in each of two subjects was canceled due to short-term illness (fever, upper respiratory symptoms).
All BRE subjects completed their exercise at an LBNP level that produced approximately one body weight for half of the bed rest period. Thereafter, as their tolerance to exercise increased, all but one of the eight subjects exercised at >1.05 times body weight. In the subject who was prone to presyncopal symptomatology, the average exercise and post-exercise LBNP were reduced to 90% body weight for part of the countermeasure period. Of the exercise sessions performed, the mean exercise time was 50 ± 2 min. Across all exercise sessions completed, the average LBNP was 52 ± 3 mmHg, which corresponded to a mean loading of 1.0 ± 0.1 body wt.
Nutritional countermeasure.
All meals were prepared for all three groups by the MEDES dietary staff, with controlled amounts of total energy and macronutrients (carbohydrate, fat, and protein) (Table 2) as well as sodium, potassium, calcium, and fluid intake. The goal of the nutrition countermeasure was to provide an additional amount of protein and free leucine during the bed rest period for the BRN group. All three groups received similar diets during the pre-bed rest period, with the protein composition maintained at 1.0 g·kg body wt–1·day–1. The BR and BRE groups continued to receive this amount of protein during the bed rest period, whereas the BRN group received 1.45 g·kg body wt–1·day–1. In addition, the BRN group received 3.6 g/day of free leucine, 1.8 g/day of free valine, and 1.8 g/day of free isoleucine equally divided out over the three meals of the day. Thus the total protein intake for the BRN group was 1.6 g/kg body wt/day. To compensate for the additional increase in energy intake from protein in the BRN group, carbohydrate content was reduced during the bed rest period. Energy intake for all three groups was adjusted downward during bed rest due to the reduced energy expenditure compared with the pre-bed rest period. Also during bed rest, the BRE group received an additional amount of energy intake equal to the energy expended during the exercise training sessions.
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A muscle biopsy (6) was obtained from the vastus lateralis of each subject before bed rest and on day 59 of bed rest (range 16–24 h after the last exercise session). The muscle biopsy was performed on day 59 to avoid interfering with other testing procedures being performed on the final day of bed rest before subject reambulation.
A portion of the sample was sectioned into several longitudinal pieces, placed in cold skinning solution (see below), and stored at –20°C for later analysis of single muscle fiber physiology. After a single muscle fiber experiment, each single fiber was analyzed for MHC composition as described below. Muscle samples were transported to the Human Performance Laboratory at Ball State University where the single muscle fiber physiology experiments were completed within a 4-wk period after the muscle biopsy.
Skinning, Relaxing, and Activating Solutions
The skinning solution contained (in mM) 125 potassium propionate, 2.0 EGTA, 4.0 ATP, 1.0 MgCl2, 20.0 imidazole (pH 7.0), and 50% (vol/vol) glycerol. The compositions of the relaxing and activating solutions were calculated with the use of an interactive computer program described by Fabiato and Fabiato (17). These solutions were adjusted for temperature, pH, and ionic strength using stability constants in the calculations (24). Each solution contained (in mM) 7.0 EGTA, 20.0 imidazole, 14.5 creatine phosphate, 1.0 free Mg2+, and 4.0 free MgATP, KCl, and KOH to produce an ionic strength of 180 mM and a pH of 7.0. The relaxing and activating solutions had a free Ca2+ concentration of pCa 9.0 and pCa 4.5, respectively (where pCa = –log Ca2+ concentration).
Single Muscle Fiber Physiology Experiments
For each experiment, a 2- to 3-mm muscle fiber segment was isolated from a muscle bundle and transferred to an experimental chamber filled with pCa 9.0 solution. Each end of the fiber was then securely fastened between a force transducer (model 400A, Cambridge Technology) and a direct current torque motor (model 308B, Cambridge Technology) as described by Moss (35). The instrumentation was arranged so that the muscle fiber could be rapidly transferred back and forth between experimental chambers filled with relaxing or activating solutions. The apparatus was mounted on a microscope (Olympus BH-2) so that the fiber could be viewed (x800) during an experiment. Using an eyepiece micrometer, sarcomeres along the isolated muscle segment length were adjusted to 2.5 µm, and the fiber length (FL) was determined. All single muscle fiber experiments were performed at 15°C.
Unamplified force and length signals were sent to a digital oscilloscope (Nicolet 310, Madison, WI), enabling muscle fiber performance to be monitored throughout data collection. Analog force and position signals were amplified (dual differential amplifier, model 300-DIF2, Positron Development, Inglewood, CA), converted to digital signals (National Instruments), and transferred to a computer (Gateway, Irvine, CA) for analysis using customized software. Servo-motor arm and isotonic force clamps were controlled with a computer-interfaced force-position controller (model 300-FC1, Positron Development).
For each single muscle fiber experiment, a fiber with a compliance (calculated as FL divided by y-intercept) >10% and/or a decrease in peak force (Po) of >10% was discarded and not used for analysis. The within-fiber test/retest results of a single muscle fiber in our laboratory for the measurements of size, force-power relationships, Po, and contractile velocity were <1%. The coefficients of variation for the force transducer and servo-mechanical lever mechanism during the 1-yr period in which we examined single muscle cell function from the women, as part of this investigation, were <1%.
Single Muscle Fiber Analysis
Individual muscle fibers were analyzed for diameter, Po, maximal unloaded shortening velocity (Vmax) (25), and force-power characteristics. Detailed descriptions and illustrations of these procedures have been previously published by our laboratory (45, 47).
Single fiber diameter. A video camera (Sony CCD-IRIS, DXC-107A) connected to the microscope and interfaced to a computer allowed viewing on a computer monitor and storage of the digitized images of the single muscle fibers. Fiber diameter was determined from a captured computer image taken with the fiber briefly suspended in air (<5 s). Fiber width (diameter) was determined at three points along the segment length of the captured image using NIH public domain software (Scion Image, release Beta 4.0.2, for Windows). Fiber diameter was calculated from the mean width of these measurements, with the assumption that the fiber forms a cylindrical cross-section when suspended in air.
Single fiber Po. The outputs of the force and position transducers were amplified and sent to a microcomputer via a Lab-PC+ 12-bit data acquisition board (National Instruments). Resting force was monitored, and then the fiber was maximally activated in pCa 4.5 solution. Active Po was determined in each fiber by computer subtraction of the baseline force from the Po in the pCa 4.5 solution.
Single fiber shortening velocity from the slack.
Fiber shortening velocity from the slack (Vo) was measured by the slack-test technique as described by Edman (16). The fiber was fully activated in pCa 4.5 solution and then rapidly released to a shorter length, such that force fell to baseline. The fiber shortened, taking up the slack, after which force began to redevelop. The fiber was then placed in pCa 9.0 solution and returned to its original length. The duration of unloaded shortening, or time between onset of slack and redevelopment of force, was determined by computer analysis. Four different activation and length steps (150, 200, 250, and 300 µm; each 
15% of FL) were used for each fiber, with the slack distance plotted as a function of the duration of unloaded shortening. Fiber Vo (FL/s) was calculated by dividing the slope of the fitted line by the fiber segment length, and the data were normalized to a sarcomere length of 2.5 µm.
Single fiber power. Submaximal isotonic load clamps were performed on each fiber for determination of force-velocity parameters and power. Each fiber segment was fully activated in a pCa 4.5 solution and then subjected to a series of three isotonic load steps. This procedure was performed at various loads so that each fiber was subjected to a total of 15–18 isotonic contractions.
For the resultant force-velocity relationships, load was expressed as P/Po, where P is the force during load clamping and Po is the peak isometric force developed before the submaximal load clamps. Force and shortening velocity data points derived from the isotonic contractions were fit by the hyperbolic Hill equation (28). Only individual experiments in which r2 was 
0.98 were included for analysis.
Fiber peak power was calculated from the fitted force-velocity parameters (Po, Vmax, and a/Po, where a is a force constant and Vmax is the y-intercept). Absolute power (µN·FL–1·s–1) was defined as the product of force (µN) and shortening velocity (FL/s). Normalized power (W/l) was defined as the product of normalized force and shortening velocity.
MHC Determination
After single muscle fiber physiology experiments were completed, each fiber was solubilized in 80 µl of 1% SDS sample buffer and stored at –20°C until assayed (50). Briefly, samples were run overnight at 4°C on a Hoefer SE 600 gel electrophoresis unit (San Francisco, CA) utilizing a 3.5% (wt/vol) acrylamide stacking gel with a 5% separating gel. After electrophoresis, the gels were silver stained as described by Giulian et al. (22). MHC isoforms were identified according to migration rate.
Whole Muscle Strength and Size
To document muscle strength changes, isometric and dynamic muscle tests were performed before and after bed rest in all three groups. MRI was conducted to determine size of the thigh muscles before and after bed rest in all three groups. Muscle strength and size procedures have been described in detail previously (3, 48).
Statistical Analysis and Calculations
Changes in MHC I and IIa single fiber variables [diameter, Po, normalized force (Po/cross-sectional area; CSA), Vo, Vmax, peak power, and normalized peak power] from pre- to post-bed rest in BR, BRN, and BRE groups were determined by two-way ANOVA with repeated measures. Only MHC I and IIa fibers were analyzed statistically because of the low number of other fiber types studied in the vastus lateralis muscle. However, data from the fiber types with relatively low yield are presented. Significance was set at P < 0.05. Post hoc analyses were conducted with a Tukey's test. All data are presented as mean values ± SE.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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The number of hybrid fibers studied after bed rest increased (P < 0.05) 23% in BR and 19% in BRN compared with the number of pure fibers (fibers consisting of a single MHC isoform) studied (Table 3). The number of hybrid fibers studied in BRE was similar (12%) before and after bed rest. Because of the small number of MHC I/IIa/IIx (n = 6) and IIx (n = 2) fibers examined throughout the study, single fiber data from these fibers were excluded.
Single Muscle Fiber Diameter
Pre- and post-bed rest single muscle fiber diameters are shown in Table 4. Before bed rest, results in the BR group showed smaller (P < 0.05) MHC I fiber diameter than in the BRN group. Bed rest reduced (P < 0.05) MHC I and IIa diameters, as shown in BR (–14 and –16%) and BRN (–19% and –21%) samples. However, in BRE samples after bed rest, MHC I or IIa diameter was unchanged. Although BRN samples showed larger MHC I fibers before bed rest, correlation analysis between initial fiber diameter and fiber atrophy (absolute change) was not significant (r2 = 0.37; P > 0.05).
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Po and Po/CSA are shown in Table 4. Before bed rest, BR samples showed lower (P < 0.05) MHC I fiber Po than BRN samples. Po was decreased (P < 0.05) 35% and 40% in MHC I fibers from both BR and BRN groups, respectively. A similar trend (P < 0.08) was observed for MHC IIa single fiber Po in both BR (–31%) and BRN (–30%) samples. For the BRE group, there were no changes in MHC I or IIa single fiber Po as a result of bed rest. When corrected for fiber size, MHC I fibers from the BR group had a 16% decline (P < 0.05) in Po/CSA after bed rest.
Single Muscle Fiber Shortening Velocity (Vo and Vmax)
Contractile velocity was assessed by both the slack-test (25) and force-velocity (Vmax) procedures (see METHODS). These measurements provided a measure of shortening velocity as shown in Table 5. There were no differences in contractile velocity from pre- to post-bed rest within the BR, BRN, and BRE groups.
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Absolute peak power and power normalized for muscle cell size are shown in Table 6. In the BR and BRN groups, absolute MHC I peak power was reduced (P < 0.05) 42% and 44%, respectively. When normalized to muscle cell size, however, these differences were not statistically significant. In the BRE group, MHC I single fiber power parameters were maintained. In the BR, BRN, and BRE groups, MHC IIa absolute peak and normalized power were unchanged after bed rest.
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Thigh muscle size was decreased (P < 0.05) by 21% in the BR and by 24% in the BRN group after bed rest. Thigh muscle power was reduced (P < 0.05) by 27% in the BR group and by 33% in the BRN group after bed rest. In the BRE group, thigh muscle size and whole muscle power were maintained with bed rest. Complete whole muscle results are presented elsewhere (48).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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To date, there have been no studies in a 1-g or microgravity environment to examine single muscle fiber contractile function with a concurrent training program. Most likely, concurrent exercise programs will be necessary to protect the various physiological systems (for example, cardiovascular, muscle, bone) of humans during long-duration space travel. The present 60-day bed rest protocol that combined resistance and aerobic exercises as a countermeasure for unloaded skeletal muscle shows that cell size and function are maintained to pre-bed rest levels. This is particularly noteworthy given the magnitude of atrophy (15%) and decline in contractile function (>40% in power) observed at the cell level in the control group. Moreover, the addition of aerobic exercise provided an adequate stimulus to maintain slow-twitch fiber size and function, without diminishing the positive benefits to the fast-twitch fibers observed in the previous bed rest study utilizing only resistance exercise (46). This is an important concept to consider because some studies investigating the effects of concurrent resistance and endurance exercise have shown an attenuation of muscle strength gains and hypertrophy compared with resistance training alone (4, 15, 27). Although no fiber hypertrophy was observed in the exercise group in the present 60-day bed rest study, the maintenance of fiber size suggests a balance between the anabolic and catabolic processes regulating muscle size (23) was achieved with the concurrent exercise program during bed rest.
As with the 90-day study utilizing resistance training in men (3), the concurrent exercise protocol used in this investigation was also effective in maintaining thigh muscle mass and power (48). In the 90-day study, this was primarily achieved by protecting fast-twitch muscle fibers and increasing the proportion of hybrid fibers containing the MHC IIa and/or IIx protein in the vastus lateralis. Although advantageous for whole muscle power, this cellular profile would lend itself to a more fatigable muscle and would favor glycolytic metabolism, neither of which would be beneficial for endurance-based activities. In the present investigation, the maintenance of whole muscle thigh characteristics with the exercise regimen was reflective of cellular size and function (strength, speed, and power) being maintained in both slow- and fast-twitch muscle fibers. This protection of muscle quantity and quality is ideal for a host of reasons ranging from crewmembers performing extravehicular activities to the metabolic health of the individual given the key role that muscle has in a variety of physiological processes.
We have previously shown that a subset of fibers used for contractile physiology measurements (15–20) is comparable to a more detailed analysis (>100 fibers) after long-term bed rest (21, 46), thus allowing for insight to MHC alterations from the present data set. The concurrent training protocol prevented an increase in the number of hybrid muscle fibers during bed rest and did not alter the proportion of pure fiber types (MHC I and IIa), as has been previously reported (46). A limited number (n = 5 fibers) of hybrid fibers expressing MHC I/IIa/IIx were observed in the present 60-day study after bed rest. This MHC isoform is typically rare; however, the number of fibers expressing MHC I/IIa/IIx increased 16% in response to 90-day bed rest (21, 46). The relatively high proportion of hybrid fibers that we have observed in two long-duration (60- and 90-day) bed rest studies is on the upper end of what has been previously reported in humans. In the skeletal muscle from inactive aging individuals (70–80 yr old), there are 35% hybrid fibers (14, 50). Only in extreme muscle unloading perturbations, such as spinal cord injuries, are more hybrid fibers and MHC IIx proteins present (33) than with long-duration bed rest. This highlights the magnitude that 2–3 mo of bed rest has in altering the MHC phenotype. Moreover, the alterations in MHC profile observed between the 60- and 90-day studies indicate that the additional 30 days of bed rest between studies (90 vs. 60 days) had a greater impact on MHC transformations.
Nutritional Countermeasure
The idea behind the nutrition countermeasure of increased protein and leucine consumption was well supported by acute muscle studies showing anabolic benefits for whole body and muscle protein synthesis (8, 9, 12, 34). Further support came from recent short-term (<30 days) bed rest studies showing positive benefits for protein turnover and lean muscle mass preservation (7, 37, 42, 43). However, the whole muscle (48) and single muscle fiber data from the present 60-day bed rest study suggest that the skeletal muscle benefits observed from acute metabolic studies and short-term bed rest studies do not extend to longer duration bed rest. Interestingly, the nutrition group lost significantly more thigh muscle mass than the control group (48). At the single fiber level, the nutrition group had a reduction in fiber diameter that was 5% greater than the control group in both MHC I and IIa muscle fibers. Although the difference in single fiber diameter between the nutrition and control groups was not significant, it does agree with the whole muscle mass data. Most likely, the additional 5% loss in fiber diameter collectively contributed to the overall loss in thigh muscle mass. On the basis of findings in the present study at the whole muscle and single fiber level, nutrition alone appears to exacerbate the loss in thigh muscle mass.
Our findings are in slight contrast to a recent study by Paddon-Jones et al. (37), who reported a benefit for leg muscle mass (using dual-energy X-ray absorptiometry) when an amino acid and carbohydrate supplementation was consumed during 28 days of bed rest. In a subgroup of subjects from the same study, vastus lateralis MHC I and IIa fiber diameters were not protected by the nutritional supplementation (19). This discrepancy between the whole muscle and single fiber data may be a product of the techniques and specificity to assess changes in muscle size, since the whole muscle measures did not isolate the thigh muscles. These authors also reported that whole muscle performance was significantly reduced in the nutrition group, although not to the same degree as in the control group (37). The offset in muscle performance between the nutrition and control groups may have been related to the elevated shortening velocity of the MHC IIa fibers (reported significant at the P < 0.1 level) relative to pre-bed rest (19). Although we did not obtain muscle biopsies during the midpoint of the 60-day bed rest, which would provide a time point for direct cellular comparison, we did find similar losses in the nutrition and control groups' thigh muscle mass (using MRI) on day 29 with a continued reduction in thigh muscle mass by day 59 of bed rest period in the present study (48).
Gender Comparisons
One of the novel aspects of this investigation was the inclusion of female volunteers. To our knowledge, potential gender differences in skeletal muscle to unloading have been relatively unexplored. Although of different study durations, the present 60-day study provides some insight to changes in skeletal muscle compared with our previous involvement in a 90-day bed rest study (46). A comparison of changes in MHC I and IIa fiber size and contractile function from the control subjects of the 60-day study (women) and the 90-day study (men) are shown in Table 7. Perhaps the most interesting aspect to note was the change in fiber size with bed rest in the men and women. The men had a greater magnitude of atrophy in the MHC I fibers (–15%) than in the MHC IIa fibers (–8%), which was in agreement with previous animal studies (18) showing a preferential atrophy of MHC I fibers during unloading. In contrast, the women had a similar degree of atrophy in both fibers types (–14 and –16%). Furthermore, the changes noted in muscle atrophy and contractile function in the women occurred with 30 fewer days of bed rest.
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Summary and Practical Applications
This study shows that the concurrent training program of resistance (2–3 days/wk) and aerobic (3–4 days/wk) exercise was protective of the thigh muscle during 60 days of bed rest in women. This finding is supported by the maintenance of whole muscle and single muscle fiber parameters (MHC I and IIa) during the bed rest period. These data indicate that, in light of the difficulty in protecting slow-twitch muscle fibers by resistance training alone during prolonged unloading (2, 3, 46), concurrent resistance and aerobic training protocols should be considered as a viable countermeasure for the upper leg of crewmembers during long-duration space travel.
This 60-day bed rest study was the first dedicated to skeletal muscle responses in females and provides a detailed profile of muscle contractile function at the cellular level. The large decline in fiber size and function in the nonexercisers was massive by clinical standards and has serious ramifications for metabolic health and mobility for individuals forced into long-term bed rest situations. The nutrition countermeasure did not provide an ergogentic benefit for single muscle fiber size and function during bed rest, which is supported by the whole muscle findings from these same women (48). This information should be useful to the international space community and the health care industry working with patients stricken with extended bed rest.
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This phase of the investigation was supported by NASA Grant NNJ04HF72G to S. Trappe and T. Trappe.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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 A. Bergouignan, G. Trudel, C. Simon, A. Chopard, D. A. Schoeller, I. Momken, S. B. Votruba, M. Desage, G. C. Burdge, G. Gauquelin-Koch, et al. Physical Inactivity Differentially Alters Dietary Oleate and Palmitate Trafficking Diabetes, February 1, 2009; 58(2): 367 - 376. [Abstract] [Full Text] [PDF]
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 E. I. Glover, S. M. Phillips, B. R. Oates, J. E. Tang, M. A. Tarnopolsky, A. Selby, K. Smith, and M. J. Rennie Immobilization induces anabolic resistance in human myofibrillar protein synthesis with low and high dose amino acid infusion J. Physiol., December 15, 2008; 586(24): 6049 - 6061. [Abstract] [Full Text] [PDF]
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N. Luden, K. Minchev, E. Hayes, E. Louis, T. Trappe, and S. Trappe Human vastus lateralis and soleus muscles display divergent cellular contractile properties Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2008; 295(5): R1593 - R1598. [Abstract] [Full Text] [PDF]
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P. A. Tesch, F. von Walden, T. Gustafsson, R. M. Linnehan, and T. A. Trappe Skeletal muscle proteolysis in response to short-term unloading in humans J Appl Physiol, September 1, 2008; 105(3): 902 - 906. [Abstract] [Full Text] [PDF]
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M. Harber and S. Trappe Single muscle fiber contractile properties of young competitive distance runners J Appl Physiol, August 1, 2008; 105(2): 629 - 636. [Abstract] [Full Text] [PDF]
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D. Slivka, U. Raue, C. Hollon, K. Minchev, and S. Trappe Single muscle fiber adaptations to resistance training in old (>80 yr) men: evidence for limited skeletal muscle plasticity Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2008; 295(1): R273 - R280. [Abstract] [Full Text] [PDF]
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 A. Bergouignan, D. A. Schoeller, S. Votruba, C. Simon, and S. Blanc The acetate recovery factor to correct tracer-derived dietary fat oxidation in humans Am J Physiol Endocrinol Metab, April 1, 2008; 294(4): E645 - E653. [Abstract] [Full Text] [PDF]
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