Periods of skeletal muscle disuse can occur for a variety of reasons, such as hospitalization that may mandate bed rest or due to injury which may require limb immobilization. The average hospital stay is 5 to 7 d when admitted with acute illness (1) and most periods of disuse requiring home-based recovery (illness or mild injury) also last less than 1 wk (2). Consequently, the clinical relevance of such short-term disuse has recently attracted considerable research attention (3–5). Studies have shown that 5 to 7 d of disuse results in marked declines in muscle size (6), muscle fiber cross-sectional area (CSA) (4), strength (3,6), and insulin sensitivity (3,7) and induces anabolic resistance of skeletal muscle tissue (8,9). This potent deconditioning effect of short-term disuse also lends support for previous suggestions that short periods of disuse may be additive over the lifespan and act as the key driver of age-related muscle loss (10,11).
To date, research on short-term disuse atrophy has mainly assessed limb or whole body fat-free mass using dual-energy x-ray absorptiometry, particularly for whole body disuse (12,13), or muscle anatomical CSA (aCSA) of large muscle groups (for example midthigh or Musculus quadriceps) using computed tomography (CT) generally when a unilateral limb immobilization model is used (6,9). Applying magnetic resonance imaging (MRI) to obtain serial images of a muscle group allows for the quantification of muscle volume of an entire muscle group, and allows for the calculation of changes in the individual constituent muscles (14,15). Furthermore, by utilizing MRI to calculate muscle volume and by assuming or calculating muscle density, the determination of muscle mass is possible (16). Therefore, MRI may provide a more sensitive tool to assess time-dependent changes in muscle mass during short periods of disuse.
In the present study, we used serial MRI scans to provide a detailed and temporal picture of upper leg muscle atrophy in combination with unilateral leg strength testing during short-term (1 wk) unilateral leg immobilization in 13 healthy, young men. We hypothesized that a decrease in thigh, M. quadriceps and M. hamstrings muscle volume would already be evident after 2 d of immobilization, and that further declines would be seen after a further 5 d of immobilization. Furthermore, we hypothesized that individual muscle groups and constituent muscles (e.g., M. quadriceps and Musculus vastus medialis and Musculus vastus lateralis) that perform more of a role in habitual physical activities would experience significantly greater reductions in muscle volume and aCSA when compared with muscles that undertake less habitual loading (e.g., the M. hamstrings and Musculus biceps femoris short head, Musculus adductor group, Musculus sartorius and M. gracilis muscles). In line with this, we hypothesized that M. quadriceps strength would decline to a greater extent than M. hamstrings strength after 7 d of unilateral leg immobilization.
Thirteen young, healthy males (age, 20 ± 1 yr; body mass index [BMI], 23.4 ± 0.9 kg·m−2; body fat percentage = 15 ± 2; lean mass = 63 ± 2 kg) volunteered to take part in this study. Prior to inclusion in the study all subjects underwent a routine medical screening to ensure no adverse health conditions were present and that they met the eligibility criteria for the study. Only young males were included in the present study since both age (17) and sex (18) can influence the rate of muscle disuse atrophy, and our goal was to maintain a homogenous population for the current study. Exclusion criteria included; a personal or family history of thrombosis, musculoskeletal/metabolic disorders (e.g., type 2 diabetes), BMI < 18.5 or >28, any musculoskeletal injury of the legs in the 12 months before starting the study, any contraindications to MRI scanning (e.g., metallic implants), participation in a structured resistance type exercise training program within 6 months prior to the study, consumption of any nutritional supplements before and during the study. During the screening visit subjects completed a general health questionnaire, body composition was assessed by air displacement plethysmography (BODPOD; Life Measurement, Inc. CA), height, weight, and blood pressure were measured. Subjects were also familiarized to the unilateral one-repetition maximum (1-RM) strength testing technique for each exercise with the dominant (i.e., stronger) leg being identified. All subjects were informed of the nature and possible risks of the experimental procedures before providing written informed consent. The study was approved by The Sport and Health Science Ethics committee of the University of Exeter (150221/B/02), in accordance with the guidelines set out in the Declaration of Helsinki. This study was registered as a clinical trial with clinicaltrials.gov (NCT02984332).
A schematic of the experimental design is shown in Figure, Supplemental Digital Content 1, http://links.lww.com/MSS/B824. After screening, eligible subjects reported to the laboratory at ~0900 h in the fasted state for a pretesting visit where 1-RM strength testing was performed. One week after the pretesting visit subjects returned to the laboratory (subjects were instructed to refrain from vigorous exercise during this period) and underwent a preimmobilization magnetic resonance imaging (MRI) scan of both thighs at ~8:00 am before starting the immobilization period at ~9:00 am. After 2 d of immobilization the subjects returned to the laboratory and underwent a second identical MRI scan at approximately 9:00 am, during which the brace was removed by the experimenter for the MRI scan and was refitted in the exact same position immediately post-MRI. The subject was assisted onto the MRI bed using a wheelchair to ensure no weight bearing of the immobilized leg occurred. After 7 d of immobilization subjects completed the final study visit, which consisted of a final MRI scan at ~9:00 am followed immediately by postimmobilization unilateral 1-RM strength testing. Subjects were only allowed to bear weight on the immobilized leg following completion of the strength testing.
The model of immobilization used was a unilateral leg brace (X-ACT Donjoy brace; DJO Global, Vista, CA, USA) with the subject ambulating on crutches (after receiving instructions) during the immobilization period. The immobilized leg was counterbalanced for leg dominance, and the nonimmobilized leg acted as a within-subject control. Using the hinge of the brace the knee was fixed at an angle of 40° flexion (i.e., full knee extension being considered as 0°) to ensure no weight bearing occurred. Subjects were instructed that all ground contact and muscle contraction (except for ankle rotation exercises twice per day to activate the venous muscle pump) in the immobilized leg were forbidden. Adhesive tape with the experimenter’s signature inscribed was placed around the straps of the brace and breaking of the tape would indicate tampering and result in exclusion from the study (17). Subjects were provided with a plastic cover to wear over the brace when showering. Daily contact was maintained with the subjects throughout the entire study to ensure compliance.
Determination of muscle volume and anatomical cross-sectional areas
A 1.5 tesla (T) MRI scanner (Intera, Phillips, The Netherlands) was used to obtain images of both thighs in the axial plane over the full length of the femur. A T1-weighted 3D turbo spin echo sequence was used (field of view, 500 × 500 mm; reconstructed matrix, 512 × 512 mm; echo time, 15 ms; repetition time, 645 ms; slice thickness, 5 mm; slice gap, 5 mm) with the subject lying still in the supine position. A four-element sense body radiofrequency (RF) coil was wrapped around both thighs. During the preimmobilization scan a specified distance from a bony landmark (femoral condoyle) on the immobilized leg in the frontal plane was used to center the axial plane images (19). This same distance was used on all subsequent MRI scans to ensure the axial images were in the same location along the length of the thigh on all scans.
Philips on-line MRI software was used to analyze the images obtained in the axial plane (the same experimenter performed all manual segmentation of the images). Starting at the most distal image (approximately mid patella), where each muscle group could be fully delineated, the muscle of interest was manually segmented to calculate slice aCSA. Moving proximally every third image was analyzed until complete delineation of the muscle groups of interest could no longer be identified (the femoral head for the M. quadriceps and thigh muscle groups and the ischial tuberosity (M. hamstrings origin) for the M. hamstrings). This was on average 16, 15 and 14 images for the thigh, M. quadriceps and M. hamstrings volume measurements, respectively (see Fig. 1 for representative images). This method for calculating muscle volume has previously been reported to give accurate and reliable results (20). We also internally validated this approach where we compared data from analyzing every image (i.e., 45 images) along the length of the muscle with the analyses of every third image (i.e., 15 images; for three subjects) which elicited equivalent calculations of muscle volume (bias, −21.6 cm3; standard deviation, 7.7; lower limits of agreement, −36.7 cm3; upper limits of agreement, −6.4 cm3). Muscle volume was calculated using a previously published method (16) where the total aCSA for all images was calculated and multiplied by the slice thickness plus the distance between slices (linear interpolation) (in this case a total of 3 cm, 5-mm slice thickness, 25-mm slice gap), summarized by the following equation:
Mid M. quadriceps aCSA was calculated by segmenting the M. quadriceps muscle in a single MR image 15 cm above the top of the patella to aid comparisons to previous studies (9,19,21). The aCSA was calculated for all the individual muscles of the thigh that could be completely delineated (11 individual muscles; M. rectus femoris, M. vastus lateralis, M. vastus intermedius, M. vastus medialis, M. semitendinosus, M. semimembranosus, M. biceps femoris short head, M. biceps femoris long head, M. adductor group (3 M. adductor muscles combined), M. sartorius, M. gracilis) of both the immobilized and control legs (muscles, such as the M. pectineus, M. psoas, and M. iliopsoas, were not analyzed as they could not be adequately visualized for quantification). The single axial MR image that had the largest aCSA for each muscle was located (at a specific location along the length of the thigh) and the muscle of interest was manually segmented as described above. This same corresponding image (on the same location of the thigh) was analyzed on scans 2 and 3 to assess for changes in aCSA over the immobilization period.
Calculations of muscle mass and muscle atrophy related to anatomical longitude
Muscle volumes calculated from the MRI scans were used to calculate muscle mass to understand the weight of muscle tissue lost during immobilization. Muscle mass was calculated by multiplying muscle volume by 1.04 g·cm−3 (the reported density of leg muscle tissue) (22) in line with previous work (16). To assess if different longitudinal sections of a muscle group atrophied to differing extents, the length of each muscle group (M. quadriceps, M. hamstrings, thigh) was first calculated by summing the thickness of all the slices and slice gaps between the most proximal and distal images analyzed. Subsequently, starting from the knee (the most distally analyzed image = 0%) the distances at 10% intervals up to the femoral head (the most proximally analyzed image = 100%) were calculated for each muscle and each subject to allow for comparisons between subjects of different heights and leg lengths. Then the aCSA analyzed at each 10% distance along the length of the muscle (or the aCSA analyzed that was closest to each 10% distance) for each muscle group and each subject was compared between preimmobilization and postimmobilization (i.e., days 0 and 7) for the immobilized leg.
Unilateral leg extension, leg curl, leg press, and calf raise exercises were performed in the stated order and for both legs separately using standard gym equipment (Life Fitness, Cambridge, UK). 1-RM strength testing was assessed using an incremental multiple repetition testing procedure that was carried out individually for each leg with the leg identified to be immobilized always being tested first. After two warm up sets of 8 and 4 repetitions at self-determined 25% and 50% of 1-RM, respectively, single repetitions at 1-RM were attempted. The weight was increased incrementally until no further weight could be lifted, with each attempt separated by a 2-min rest. The final 1-RM lift was taken as the heaviest repetition that was successfully completed with correct technique where full range of motion was achieved.
All data are presented as means ± SEM, and all statistical analyses were conducted in GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA). Two-way repeated-measures ANOVA with leg (immobilized and control) and time (pre and post (for strength); or day 0, day 2, and day 7 (for MRI)) as within subjects factors were used to compare differences in strength, muscle volume, and aCSA. For the immobilized leg only, a two-way ANOVA with muscle group and time as within subject factors was used to assess if different muscle groups responded differently to immobilization. Pearson’s correlation analyses were used to assess the relationship between initial muscle size (M. quadriceps and M. hamstrings) or preimmobilization muscle strength (leg extension and leg curl) with the absolute amount of muscle volume lost after 7 d for each muscle group. A Pearson’s correlation was also used to assess the relationship between preimmobilization aCSA along the longitude of each muscle (thigh, M. quadriceps, and M. hamstrings) and the aCSA lost along the longitude of each muscle after 7 d of immobilization. For all ANOVA, when a significant interaction was found Bonferroni post hoc tests were applied to locate individual differences. Statistical significance was set at P < 0.05.
Muscle group volumes and mass
Muscle volume of the thigh, M. quadriceps, and M. hamstrings muscle groups as computed from MRI scans are displayed in Figure 2. There were no differences in muscle volume between the legs preimmobilization for any of the muscle groups ((thigh: control leg = 4651 ± 222 cm3, immobilized leg = 4697 ± 237 cm3 (P > 0.05); M. quadriceps: control leg = 2315 ± 120 cm3, immobilized leg = 2342 ± 129 cm3 (P > 0.05); M. hamstrings: control leg = 833 ± 40 cm3, immobilized leg = 830 ± 38 cm3 (P > 0.05)). In the control leg muscle volume remained unchanged at all time points in all muscle groups (all P > 0.05). Significant time–leg interactions were detected for thigh (P < 0.001), M. quadriceps (P < 0.001) and M. hamstrings (P < 0.001) such that the immobilized leg thigh muscle volume decreased by 1.7% ± 0.3% (to 4617 ± 236 cm3, P < 0.001), comprising a 1.7% ± 0.3% decline in M. quadriceps volume (to 2301 ± 127 cm3; P < 0.01) and 1.4% ± 0.2% decline in M. hamstrings muscle volume (to 818 ± 37 cm3; P < 0.001)) after 2 d. Similarly, after 7 d of immobilization thigh, M. quadriceps and M. hamstrings muscle volumes had decreased by a further 3.9% ± 0.4%, 5.0% ± 0.6%, and 2.1% ± 0.2%, respectively (to 4438 ± 223, 2186 ± 117 and 801% ± 37 cm3, respectively). This resulted in a total muscle volume loss of 5.5% ± 0.6%, 6.7% ± 0.6% and 3.5% ± 0.3% from thigh, M. quadriceps, and M. hamstrings, respectively, over the entire week. The contribution of M. quadriceps, M. hamstrings, and remaining (e.g., M. adductor group, M. sartorius, M. gracilis) muscle volume lost to total thigh muscle volume lost after 7 d is shown in Figure 3A. The relative loss of muscle volume was not different between the M. hamstrings and M. quadriceps during the first 2 d of immobilization, but was significantly greater in the M. quadriceps between 2 and 7 d (P < 0.01) and over the entire week (P < 0.001). When expressed as muscle mass, similar changes in the leg muscles were observed with immobilization. Specifically, there were no differences in thigh (4837 ± 231 g and 4885 ± 246 g; P > 0.05), M. quadriceps (2407 ± 124 g and 2435 ± 134 g; P > 0.05) or M. hamstrings (866 ± 41 g and 863 ± 40 g; P > 0.05) muscle mass between control and immobilized legs, respectively, and no significant changes were observed with time in any of the muscle groups in the control leg. In the immobilized leg thigh, M. quadriceps, and M. hamstrings muscle groups lost 83 ± 14 g (P < 0.01), 42 ± 8 g (P < 0.01) and 12 ± 2 g (P < 0.001) of muscle mass, respectively, after 2 d of immobilization. After 7 d of immobilization the immobilized leg had lost 269 ± 32 g from the thigh muscle (P < 0.001), which was attributed to a M. quadriceps muscle mass loss of 162 ± 19 g (P < 0.001) and M. hamstrings muscle mass loss of 30 ± 3 g (P < 0.001). After 7 (day 7 M. quadriceps to M. hamstrings mass difference = 132 g, P < 0.001) but not 2 (day 2 M. quadriceps to M. hamstrings mass difference 30 g, P > 0.05) days of immobilization the M. quadriceps lost more muscle mass than the M. hamstrings.
Mid-M. quadriceps and individual muscle aCSA
There were no differences in mid-M. quadriceps aCSA at the preimmobilization timepoint (control leg = 7940 ± 306 mm2, immobilized leg = 8068 ± 326 mm2 (P > 0.05)). In the control leg mid- M. quadriceps aCSA remained unchanged at all time points (all P > 0.05). Significant time–leg interactions were detected for mid-M. quadriceps aCSA (P < 0.001) such that the immobilized leg mid- M. quadriceps aCSA decreased after 2 d by 1.6% ± 0.3% (to 7935 ± 331 mm2, P < 0.05) and 7 d by 6.3% ± 0.5% (to 7556 ± 300 mm2, P < 0.001).
Individual muscle aCSA for the control and immobilized legs during immobilization are presented in Figure 4A and B. For all the individual muscles, there were no differences between the control and immobilized leg preimmobilization (P > 0.05). No changes were observed in any individual muscle aCSA during immobilization in the control leg (P > 0.05) with the exception of the adductor muscle group which decreased after 7 d (1.3% ± 0.5%, P < 0.05). Significant time–leg interactions were found for all individual muscles (all P < 0.001 except adductor group P < 0.01) except the sartorius (P = 0.056) and gracilis (P > 0.05). After 2 d of immobilization the following individual muscles’ aCSA decreased; M. vastus lateralis by 2.6% ± 0.4% (P < 0.001), M. vastus medialis by 2.0% ± 0.5% (P < 0.01), M. vastus intermedius by 2.5% ± 0.4% (P < 0.05), M. rectus femoris by 1.4% ± 0.4% (P < 0.05), M. semitendinosus by 1.7% ± 0.5% (P < 0.01), and M. adductor group by 1.4% ± 0.4% (P < 0.05). After 7 d of immobilization all individual muscles except the M. gracilis and M. sartorius had decreased in the immobilized leg; M. vastus lateralis by 7.2% ± 0.8%, M. vastus medialis by 7.0% ± 0.7%, M. vastus intermedius by 6.9% ± 1.2%, M. rectus femoris by 4.5% ± 0.6%, M. biceps femoris long head by 5.4% ± 0.6%, M. biceps femoris short head by 2.9% ± 0.6%, M. semimembranosus by 5.0% ± 0.6%, M. semitendinosus by 4.6% ± 0.6%, M. adductor group by 3.5% ± 0.5% (all P < 0.001). The contribution of each of the 11 individual muscle CSA lost to total thigh muscle CSA lost after 7 d is shown in Figure 3B. There were differences in relative muscle loss between individual muscles over the entire week of immobilization (P < 0.001). Specifically, the M. vastus lateralis lost significantly more relative aCSA than the M. adductor group (P < 0.05), M. biceps femoris short head (P < 0.01), M. sartorius (P < 0.001) and M. gracilis (P < 0.001). The M. vastus intermedius reduced in aCSA to a greater degree than the M. biceps femoris short head (P < 0.05), M. sartorius (P < 0.01) and the M. gracilis (P < 0.001) muscles. The M. vastus medialis lost significantly more aCSA compared with the M. biceps femoris short head (P < 0.01), the M. sartorius (P < 0.01) and the M. gracilis (P < 0.001) muscles.
Muscle atrophy related to anatomical longitude
The data presented in Figure, Supplemental Digital Content 2, show the absolute and relative muscle volume for the thigh (a and b), M. quadriceps (c and d) and M. hamstrings (e and f) muscle groups for the immobilized leg expressed as declines in aCSA along the length of the muscle (i.e., separated into 10% segments from knee to hip), http://links.lww.com/MSS/B825. After 7 d of immobilization the thigh, M. quadriceps and M. hamstrings experienced heterogenous absolute reduction in aCSA along the length of the muscle (Figure, Supplemental Digital Content 2a, c and e, http://links.lww.com/MSS/B825 all P < 0.001) which was still the case for the thigh and M. quadriceps when expressed as relative loss (P < 0.001), but not the M. hamstrings (P > 0.05) (Figure, Supplemental Digital Content 2b, d, and f, http://links.lww.com/MSS/B825). For the thigh, the central portion of the muscle (middle 10%, 50%–60%) lost aCSA to a greater absolute extent (50% = 972 ± 139 mm2 and 60% = 1141 ± 144 mm2) compared with the most proximal 10% (90%–100% of thigh length, hip joint. 90% = 335 ± 126 mm2 and 100% = −28 ± 82 mm2) and the most distal 10% (0%–10% of thigh length, knee joint. 0% = −1 ± 21 mm2, 10% = 84 ± 103 mm2) sections of the thigh muscle. All 10% increments from 30% to 70% along the length of the thigh muscle reduced in aCSA in comparison to 0% (all P < 0.001 except 30% compared with 0%, P < 0.05) and 100% (all P < 0.001, except 30% compared to 100%, P < 0.01) where no change in aCSA was found between 0 and 7 d of immobilization (P > 0.05). When looking at the relative change in aCSA along the length of the thigh muscle, the only differences were 50% (−6.0% ± 0.8%, P < 0.05) and 60% (−6.5% ± 0.7%, P < 0.05) sections which declined to a greater extent than the 0% section (1.1% ± 2.5%). Similar to the thigh, the M. quadriceps also experienced largest absolute reduction in aCSA in the central portion of the muscle (Figure, Supplemental Digital Content 2c, http://links.lww.com/MSS/B825), where 40% to 70% area above the knee reduced in aCSA significantly more than 0% and 100% (all P < 0.001). Although there were fewer significant differences in relative M. quadriceps decreases in aCSA (Figure, Supplemental Digital Content 2d, http://links.lww.com/MSS/B825), again the central area of the M. quadriceps (40%–70%) decreased in aCSA more than 0% (P < 0.05) but not 100% (P > 0.05). In contrast, the M. hamstrings experienced less variation in absolute reductions in aCSA (Figure, Supplemental Digital Content 2e, http://links.lww.com/MSS/B825) where only 40% and 50% areas above the knee declined more than 0% and 100% (both P < 0.05), and no differences were found along the length of the M. hamstrings muscle when relative declines in aCSA was assessed (Figure, Supplemental Digital Content 2f, http://links.lww.com/MSS/B825).
Unilateral 1eg strength data are presented in Table 1. Maximum strength decreased for leg extension (by 18.7% ± 1.8%, P < 0.001), leg press (by 21.0% ± 3.5%, P < 0.001) and calf raises (by 8.3% ± 1.5%, P < 0.001) in the immobilized leg, with no changes in the control leg. Neither the immobilized nor control leg changed in maximum strength for the leg curl exercise after 7 d of immobilization (P > 0.05).
Correlational analyses are shown in Figure 5. Preimmobilization muscle volume showed a moderate but significant negative correlation with the absolute (but not relative) amount of muscle volume lost for both the M. quadriceps (absolute, r = −0.679, P < 0.01; relative, r = −0.15, P > 0.05) and M. hamstrings muscle groups (r = −0.592, P < 0.05; relative, r = −0.10, P > 0.05), with the thigh muscle showing a trend for the same response (r = −0.501, P = 0.081; relative, r = −0.06, P > 0.05). The absolute change in M. quadriceps volume had a moderate positive correlation with the absolute change in leg extension strength (r = 0.563, P < 0.05), though no significant correlation was observed between the absolute loss of M. hamstrings volume and absolute change in leg curl strength after 7 d of immobilization (r = −0.592, P < 0.05). Preimmobilization aCSA along the length of the thigh, M. quadriceps and M. hamstrings muscles all had a positive correlation with aCSA lost along the length of each muscle respectively after 7 d of immobilization (thigh, r = 0.718 P < 0.001, M. quadriceps, r = 0.741 P < 0.001, M. hamstrings, r = 0.596 P < 0.001) (Fig. 5).
The present study demonstrates that thigh muscle atrophy occurs rapidly (within 2 d) following the onset of muscle disuse (limb immobilization via leg brace) and continues at a similar rate for at least 1 wk (by approximately 1% per day). The majority of thigh muscle loss during 1 wk of immobilization was mainly attributed to the loss of M. quadriceps volume rather than M. hamstrings volume, and was reflected by a greater decline in leg extension compared with leg flexion strength. Of the 11 individual thigh muscle groups, the M. vastus lateralis was the most susceptible to short-term disuse atrophy.
The study of short-term (< 1 wk) muscle disuse atrophy has direct applications to clinical practice and rehabilitation where physical inactivity is mandated (21,23). Here, we report a detailed characterization of thigh muscle atrophy during 1 wk of leg immobilization. We report a basic rate of upper leg muscle atrophy over 1 wk in line with previous comparable studies. For example, we observed thigh muscle aCSA at the mid-part of the upper leg (15 cm above the patella) atrophied by ~6%, largely explained by a ~6% loss of M. quadriceps aCSA (with only a ~3% loss of M. hamstrings aCSA), which is in keeping with previous reports of upper leg muscle atrophy (generally M. quadriceps only) determined by single slice CT scan during 1 wk of leg immobilization (leg cast) (i.e., ~6% atrophy) (24,25). The use of MRI in the present study also allowed us to extend on these findings by taking multiple images (~45, of which ~15 were manually segmented) across the whole upper leg allowing us to report thigh muscle, M. quadriceps and M. hamstrings volumes temporally across 1 wk of unilateral leg immobilization. This approach allowed the striking observation that muscle volume of the thigh, M. quadriceps and M. hamstrings had already decreased substantially (by 1.7% ± 0.3%, 1.7% ± 0.3%, and 1.4% ± 0.2%, respectively) following merely 2 d of immobilization. The M. quadriceps had atrophied to a greater extent (6.7% vs 3.5%) when compared with the M. hamstrings after 7 d (see Fig. 2B and C). Given that the M. quadriceps muscle comprises the largest muscle group of the upper leg, it is clear that M. quadriceps atrophy largely explains thigh disuse atrophy during short term immobilization (see Fig. 3A). Indeed, the susceptibility of the M. quadriceps to atrophy to a greater extent than the M. hamstrings could, at least in part, be explained by its larger mass (9,15). In support, we report both initial M. quadriceps and M. hamstrings volumes (Fig. 5B and C), as well as the larger longitudinal areas of the muscle (Fig. 5E and F), correlate with the volume of muscle lost and the aCSA lost along the longitude of each muscle, after 7 d of immobilization, respectively. However, it is also likely that the degree of atrophy experienced by the two constituent muscle groups of the thigh is also influenced by other factors such as the brace model utilized, where the M. quadriceps are immobilized at a longer length (of the muscle fascicles and overall muscle length, e.g., distance between origin and insertion) than the M. hamstrings. Previous research has shown that the length at which a muscle is immobilized affects its rate of atrophy (26), which also impacts upon its basal tone throughout disuse (27) which may also modulate the rate of muscle atrophy. Furthermore, other factors such as differing muscle fiber composition (28,29), differing “training” status (i.e., habitual gravitational loading) (30), or differential expression of regulatory genes/proteins across muscle groups (28,31), may contribute to the differential atrophy observed across muscle groups. Worthy of note is that this divergent disuse atrophy response of the M. quadriceps and M. hamstrings may be a transient phenomenon given that longer term (14 d) studies (albeit during bedrest) do not report any differences between the rate of M. quadriceps (−6.5%) and M. hamstrings volume (−6.1%) decline (32).
We also used our muscle volume measurements to estimate muscle mass. This allowed for the quantification of actual muscle tissue lost as a result of disuse, which amounted to 83 g from the thigh muscle after 2 d of immobilization (M. quadriceps = 42 g and M. hamstrings = 12 g). Over the full week of disuse the thigh muscles atrophied by 269 g, consisting of 162 g loss specifically from the M. quadriceps and 30 g from the M. hamstrings. Interestingly, these measurements slightly exceed previous estimations extrapolated from CT approaches (33) which report ~220 g lost from a whole leg, or estimated changes of mass based on alterations in muscle protein turnover from stable isotope experiments which have estimated 150–200 g (9,21). These data highlight the rapid and substantial muscle mass loss consequent with disuse, and underlines the importance of understanding the underlying mechanisms and the development of effective interventions (34–36), for combatting functional and metabolic decline in multiple populations.
The resolution of MRI allows for the assessment of the aCSA of the individual muscles of the thigh, M. quadriceps and M. hamstrings. In this respect, we present the first full assessment of how each of the constituent individual muscles of the thigh temporally respond to 1 wk of immobilization (see Fig. 4A and B). The rapid muscle disuse atrophy seen at 2 d of immobilization at the level of the whole thigh was detectable by the majority of the individual muscles, with the exceptions of the M. biceps femoris long head, M. biceps femoris short head, M. semimembranosus, M. gracilis, and M. sartorius (see Fig. 4B). After 7 d of immobilization, only the M. gracilis and M. sartorius did not exhibit decreased aCSA. The M. biceps femoris short head, M. adductor group, M. sartorius, and M. gracilis typically atrophied to a lesser extent than the M. vastii muscles (Fig. 4B). These data are in line with previous (longer term) bed rest studies (15,37) which also report that the M. gracilis and M. sartorius are more resistant to disuse atrophy, though this has not been seen in other studies (38); however, due to the complexity of bed rest studies, a limited number of participants (n = 6) were included. Pooling data from multiple bed rest or leg immobilization studies may lead to better determination of possible atrophy-resistant muscle groups (particularly of smaller muscle groups e.g., M. gracilis). Overall evidence suggests that the M. gracilis and M. sartorius are more resistant to atrophy. This is likely to be due to the low gravitational load placed upon these muscles (30) and the low activation of these muscles during common habitual activities, for example, walking (39).
The heterogeneous rates of muscle disuse atrophy that we observed throughout the thigh were reflected by divergent effects on muscle strength when activating those muscle groups. Specifically, leg extension strength, during which the force generation is provided almost exclusively by the activation of the M. quadriceps, declined considerably more when compared with the leg curl (where the M. hamstrings is the primary agonist) (Table 1). This mirroring supports the contention that the rate of muscle loss is a primary factor determining the loss of function during disuse (17). Indeed, we also observed a correlation between the amount of mass lost from the M. quadriceps and the magnitude of functional decline (Fig. 5G). Though not determined in the present study, it is also clear that such a decline in muscle strength would also impact on muscle power, as has previously been noted (17). This provides an additional challenge specifically for the injured athlete when considering the multifactorial rehabilitation that may be required to return to full functional capacity. Though previous research has repeatedly shown unilateral leg immobilization results in a reduction in M. quadriceps strength (17,21,40), fewer studies have assessed the impact on M. hamstrings strength. However, 1 wk of bed rest has been shown to reduce M. quadriceps and M. hamstrings strength (isometric MVC) to a similar degree (12%–13%) (41) implying that our data showing M. hamstrings to be remarkably resistant to disuse induced declines in function may be attributable to the model of disuse which necessitates that the two muscle groups are immobilized at different positions (e.g., M. quadriceps at a longer length than the M. hamstrings).
The rapid (i.e., within 2 d) and muscle (group) specific decline in mass and function that we report during immobilization has important practical implications. For example, following a sports injury or in a more clinical setting, these data should encourage practitioners to begin appropriate interventions (e.g., neuromuscular electrical stimulation (NMES) (34,43) or higher protein intakes; to counteract muscle decline as soon as it is safe to do so. If an injury or illness is sustained and a subsequent period of non–weight-bearing is mandated, interventions should focus on larger muscle groups (e.g., M. quadriceps) as these are more susceptible to atrophy and functional decline (partly due to greater habitual gravitational loading). In immobilization (not bed rest) the M. quadriceps seems uniquely resistant to declines in function with the M. hamstrings being resistant after 1 wk. Such interventions may be beneficial since the data (43) indicate that reducing muscle loss and the metabolic decline of muscle tissue during disuse will aid in a faster return to preinjury strength level and return to play in sports (43). A limitation of the present study is that only males were included. Although this allowed for a more homogenous population to allow us to precisely measure and describe temporal, muscle specific disuse atrophy, it prevents the data being generalizable to females. Given that sex-based differences in the rate of muscle disuse atrophy likely exist (18), it is of importance that future work assesses whether similar results are demonstrable in females.
In conclusion, we report in this study that thigh muscle atrophy occurs rapidly (within just 2 d) and at a sustained rate (by approximately 0.8% per day) during 1 wk of immobilization, and this atrophy is mainly attributed to the loss of M. quadriceps tissue mass. Furthermore, the constituent muscles of the thigh atrophy during immobilization at markedly differing rates (~0.4%–1.0% per day). The preponderance toward M. quadriceps rather than M. hamstrings atrophy during immobilization is accompanied by functional declines manifesting distinctly within leg extension movements.
The authors would like to thank the Royal Society, Exeter University and Maastricht University who all contributed funding to support the completion of this work. Jonathan Fulford’s salary was supported via an NIHR grant to the University of Exeter (CRF/2016/10027).
Conflicts of interests: No conflicts of interest, financial or otherwise, are declared by the authors. The results of the present study do not constitute endorsement by the American College of Sports Medicine. The results of the study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation.
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