Human muscle atrophy results from muscle disuse, for example, bed rest (1,4,7–9,24), the unloading of a limb by suspension (6,13), or the microgravity environment during a space flight (10,17,24). Most recent studies examining changes in lower limbs have noted remarkable deterioration in both muscle morphology and function from muscle disuse (1,2,4,6–10,13,17,24). Furthermore, many studies have been done to establish effective countermeasures for the space flight environment (4,10). However, the lower-limb muscles are mostly weight bearing, and therefore the positioning and volume of these muscles are different from those in the forearm. Lower-limb muscles are likely more affected by the microgravity environment than those of the forearm. Although the forearm may be involved in many important activities in a microgravity environment, few studies have determined the extent of forearm muscle atrophy by disuse (18,19,23). Therefore, it seems very important to evaluate muscle structural and functional changes in the forearm, and also to verify effective countermeasures to prevent those changes. It has been reported that 2 wk of microgravity environment is long enough to produce muscle atrophy in the lower limbs (2), although the reduction rate of the cross-sectional area (CSA), muscle volume, maximum muscle strength, and the fatigabilities are not always parallel (1,2,4,6–10,13,17–19,23,24). The forearm may go through a different process of atrophy compared with lower limbs. To investigate this process we selected a 21-d forearm immobilization model, a period long enough to produce muscle changes in the lower limbs.
Muscle strength, torque, and fatigability as indicators of muscle function have been measured in many studies (2,4,6,7,10,13,17–19,23,24). However, only a few studies have measured the changes of muscle oxidative capacity by the use of biopsy specimens (7,9,10,18,19,24).
We estimated muscle oxidative capacity using 31phosphorous magnetic resonance spectroscopy (31P-MRS), as well as grip strength and grip endurance as indications of muscle function. As for the muscle morphology, we measured the forearm CSA and circumference. Therefore, the purpose of this study was to examine whether 21-d forearm immobilization produces changes in muscle morphology and function.
Six healthy men (age: 21.5 ± 1.4, mean ± SD) volunteered to participate in this study. All subjects were physically active, but none participated in regular training programs requiring forearm exercise before or during the study. They had previously given their written informed consent. This study was approved by the Ethics Committee of National Space Development Agency of Japan (NASDA).
Immobilization of the forearm.
The nondominant arm was immobilized for 21 d with a cast (CAST), and the dominant arm was measured as control (CONT). The cast was placed from the mid-biceps region of the upper arm to the mid-palm region of the hand. The encasted elbow and the wrist were set in a natural position, with the cast suspended from the neck by a sling. Subjects were instructed to wear the sling in the daytime, except when changing cloths and bathing. They were unable to remove the cast by themselves during the study.
Morphological and functional indicators.
Muscle morphology measurements included forearm CSA using MR imaging (MRI) and maximum forearm circumferences using tape measurement. Muscle function measurements included muscle oxidative capacity determined by noninvasive 31P-MRS measurement, maximum grip strength determined by dynamometer, and grip endurance determined by hand grip ergometer (11). All measurements were conducted before (PRE) and after (POST).
Forearm circumference and muscle CSA measurement.
Forearm circumference was measured at the maximum point by the same researcher. The selected CSA point was 5 cm distal from the inside elbow joint, which is the middle of the finger flexor muscle area. Subjects were imaged in a supine position with the arm extended. The arms were relaxed and positioned straight at the side of the body with the palms facing up. The MRI was performed with a 0.3 T to measure forearm muscle CSA (Airis, Hitachi Medico). Transverse scans were carried out with an interplaced gap of 0 mm from the elbow joint to the wrist. Spin-echo, multislice sequences with a thickness of 10 mm were used with a repetition time (TR) of 250 ms and an echo time (TE) of 25 ms.
The perimeters of the musculature, the radius, and the ulna were traced by the same researcher, double blind. The traced images were transferred to a Macintosh computer (power Macintosh G4) for calculation of the CSA using a public-domain National Institutes of Health (NIH) image software package. The CSA of the forearm muscle was defined as the enclosed musculature area minus the areas of the radius and the ulna (Fig. 1).
Muscle oxidative capacity measurement.
A 31P-MRS (BEM250/80, Otsuka Electronics Inc.) with a 2.0-T superconducting 26-cm bore magnet was used to measure 31P spectra. A double-tuned (1H and 31P), 3-cm-diameter circular surface coil was used for forearm finger flexor muscle measurement. The kinetics of PCr, inorganic phosphate (Pi), and intramuscular pH were simultaneously measured by 31P-MRS. MRS spectra were obtained every 10 s. The areas and positions of β-ATP, PCr, and Pi peaks were determined by a nonlinear curve fitting. Then, PCr and Pi peak areas were quantified from the ratio of PCr and Pi areas to β-ATP area. As there were no data available for direct chemical analysis of forearm muscle, the ATP concentration reported from muscle biopsies of the human vastus lateralis (ATP = 8.2 mM) was used for PCr quantification for measuring resting forearm PCr level (11). The muscular pH was calculated from the median chemical shift of the Pi peak relative to PCr (16).
Each subject sat on a chair with his forearm positioned inside the magnet. The elbow was naturally extended, and the handgrip on the ergometer handle was adjusted so the subjects could grip the handle comfortably. The subjects then performed handgrip isotonic contractions at a frequency of one contraction every 4 s (0.25 Hz) for 1 min. The ergometer mass was adjusted to 40% maximum muscle strength as measured in the preimmobilized period.
Muscle oxidative capacity was evaluated by time constant (Tc) for PCr recovery (PCr-Tc) after submaximal exercise (5,15). The kinetics of PCr resynthesis can be described by the following equation:EQUATION
where PCr is the PCr concentration at time t during recovery, PCr0 is the PCr concentration at the end of exercise, ΔPCr is the amount of PCr concentration during recovery, k is the rate constant of the mono-exponential curve, and 1/k is the Tc.
Maximum forearm grip strength measurement.
The hand dynamometer was used to measure forearm maximum grip strength at PRE and POST. Maximum grip strength was measured two to three times with 10-min rest between each measurement. The maximum datum was used as the maximum grip strength.
Grip endurance measurement.
Each subject performed handgrip isotonic contractions by using handgrip ergometer at a frequency of one contraction every 1 s (1 Hz) until exhaustion. The subjects lifted a mass a distance of 2 cm adjusted to 30% of maximum muscle strength. As the maximum grip strength decreased after the immobilization, two intensities were used: 30% of premaximum grip strength (absolute load) and 30% of the day-maximum grip strength (relative load) for the CAST. The ergometer was set up inside the 31P-MRS magnet to measure the kinetics of PCr and intramuscular pH during the grip endurance exercise. The end point of the exercise was judged when the subjects could not keep up with a metronome pitch set at 1-s intervals. This endpoint was recorded as the grip endurance, expressed in number of handgrip contractions.
Statistical analysis was performed by paired Student’s t-test for the data in forearm circumference and CSA. Paired Student’s t-test and unpaired Student’s t-test were used to investigate the changes in grip strength, muscle oxidative capacity, and grip endurance. Data are presented as means ± SD. A P value less than 0.05 was considered to indicate a statistically significant difference.
After the 21-d forearm immobilization, forearm CSA showed no significant change from PRE to POST in either the CAST (40.9 ± 2.6 cm2 and 40.6 ± 2.5 cm2) or the CONT (42.9 ± 4.0 cm2 and 41.0 ± 4.2 cm2) (Fig. 2). Forearm circumference showed no significant change from PRE to POST in either the CAST (24.7 ± 0.6 cm and 24.6 ± 0.7 cm) or the CONT (25.1 ± 1.0 cm and 25.1 ± 0.8 cm) (Fig. 2). PCr-Tc was significantly delayed by immobilization, from 35.0 ± 4.2 s to 62.9 ± 13.4 s for CAST. On the other hand, the PCr-Tc showed no change in from PRE (38.4 ± 9.2 s) and POST (39.7 ± 6.8 s) for CONT (Fig. 3). No change was seen in the resting forearm PCr level from PRE to POST in both CAST: 33.3 ± 4.1 mM and 35.0 ± 2.5 mM, and CONT: 33.1 ± 3.1 mM and 34.8 ± 4.5 mM. Maximum grip strength was significantly decreased by immobilization, CAST: 411.6 ± 34.6 N and 338.1 ± 33.1 N. No significant change was observed in the CONT: 433.7 ± 32.1 N and 401.8 ± 55.4 N (Fig. 4). Grip endurance at the absolute load was decreased for CAST from PRE to POST: 49.8 ± 5.9 times to 40.3 ± 5.6 times. No significant change was seen in the CONT: 47.5 ± 11.6 times and 47.2 ± 5.6 times (Fig. 5). Grip endurance at the relative load showed no significant change from PRE to POST in the CAST: 49.8 ± 5.9 times and 47.5 ± 11.3 times (Fig. 5). No difference was observed between PRE and POST in the rates of change of pH and PCr during the grip endurance exercise for both absolute and relative loads for CAST. At the end point of the grip endurance exercise, both pH and ΔPCr showed no change at the absolute load or at the relative load: for absolute load pH: PRE (6.5 ± 0.3) and POST (6.6 ± 0.1), ΔPCr: PRE (13 ± 2.8 mM) and POST (14 ± 3.1 mM), for relative load pH: PRE (6.5 ± 0.3) and POST (6.6 ± 0.2), ΔPCr: PRE (13.4 ± 2.8 mM) and POST (13.0 ± 2.4 mM).
A new major finding in the present study is the remarkable reduction in muscle function after 21-d forearm immobilization. This reduction was indicated by muscle oxidative capacity as determined by PCr recovery rate (20) (decreased by 45% on average), maximum muscle strength (decreased by 17.9% on average), and the grip endurance at the absolute load (decreased by 19.1% on average). However, there was no change observed in evaluated morphology. Also, this is the first study that noninvasively measures the change in oxidative capacity due to immobilization in the forearm. The limitations of this forearm immobilization model as a simulation of microgravity environment are that the model prohibits elbow joint movement and that the other functional changes of the whole body may not be taken into account. However, it may worth comparing the data with the other studies of simulated microgravity environment or with the data from the space flights and discuss about the differences in those changes.
Because we did not perform analysis of muscle biopsy samples or calculate muscle volume, MRI analysis of forearm muscle CSA provides information for just a single morphological change. Furthermore, as there are few studies that have measured forearm muscle morphological change by immobilization, it is necessary to compare our data with the other data of muscle immobilization studies. In a past study using 9-d forearm immobilization, CSA decreased 4.1% (4). No change (decreased 0.04% on average) was seen in our study. In past studies measuring CSA in a lower limb, CSA decreased 12% in the thigh muscle after 6 wk of unilateral lower-limb suspension (6). After 30-d bedrest, CSA decreased 8.1% in the thigh muscle and 4.8% in the calf muscle (7). LeBlanc et al. (17) reported a 6.0, 8.0, and 6.3% decrease in quadriceps femoralis, hamstring, and calf muscle CSA, respectively, after an 8-d space flight. After a study of 20 d of bed rest, muscle thickness, which is significantly correlated with muscle CSA, decreased 13% for the thigh muscle. No changes were observed in the triceps brachii muscle (1). Several studies have noted a greater effect of disuse on postural and antigravity muscles than on phasic muscles (10,13,24). In addition, the extent of atrophy was inversely related to preunloading fiber size (10). In a past study, circumference of the upper arm decreased an approximate 5% after 5 wk of immobilization (19). However, no change (decreased 0.34% on average) was seen during our study. This difference in rate may be due in part to differences in the immobilized period of time (5 wk compared with 21 d) or in the measuring site of the arm (upper arm vs forearm). In addition, in order not to cause muscle stiffness, we allowed the subjects to move their fingers during the immobilization period. There is a possibility that the muscle morphology might change if the fingers were not immobilized.
The 21-d immobilization may not have been long enough to change forearm muscle morphology. Another possibility is that changes in forearm muscle morphology may have been masked by the muscle edema effect. In further studies, we must determine whether the forearm muscle morphology will change due to longer immobilization.
Muscle oxidative capacity.
McCully et al. (20) demonstrated that PCr-Tc is significantly correlated with mitochondrial oxidative enzymes as measured with biopsy specimens. They also showed that the PCr-Tc were slower in the older subjects than in the younger and slower in subjects with chronic fatigue syndrome than in the normals (21,22). Therefore, for this research, we used PCr-Tc as a reliable indicator to estimate the muscle oxidative capacity. When the muscle oxidative capacity decreases by immobilization, the PCr-Tc will be delayed. In this study, the rate of PCr recovery (1/Tc) decreased from 0.029 to 0.016 for the CAST (reduced by 45%).
In a past study using biopsy specimens, after 42-d bed rest, calculated volume density of total muscle mitochondria decreased by 17%, muscle enzyme activities decreased by 9–11%, and the total capillary length decreased by 22.2% in the thigh muscle (9). After 5 wk of arm immobilization, PCr and glycogen concentrations in the triceps brachii were reduced by 25% and 40%, respectively (19). These findings suggest that the muscle disuse may cause a change in the source of energy supply.
In this study, no change was seen for the resting forearm PCr level calculated by 31P-MRS, although we found that the PCr-Tc was significantly prolonged. The PCr-Tc may be delayed by the changes of the muscle oxidative capacity, such as the changes of the mitochondrial volume and numbers, oxidative enzyme activities, and muscle fiber types. However, the evaluated rate of oxidative capacity reduction was much higher than the rates found in past studies using biopsy specimens (9,19). As no biopsy was performed, we did not investigate changes of mitochondrial volume, oxidative enzyme activities, muscle fiber types, and capillary length. Keller et al. (14) demonstrated by using 31P-MRS that Tc for PCr recovery after exercise may be affected by the degree of arterial stenosis and the muscle blood flow. PCr recovery was slower in patients with occlusive arterial disease of the legs than in normals.
In the 9-d forearm immobilization study, concentric wrist flexion and extension decreased by 19.9% and 32.5%, respectively (23). In the 5-wk arm immobilization study, maximal elbow extension strength decreased by 41% (18). In the 6-wk unilateral lower-limb suspension study, concentric knee extension peak torque decreased by 19.9% (13). In our study, average maximum grip strength decreased significantly by 17.9%. It is well known that with training, muscle strength increases in proportion to the increase of muscle CSA and decreases inversely when training is stopped (18,19). However, no significant CSA change was seen in this model. This dissociation between the loss of muscle strength and muscle size has been shown in previous studies (6,10,18,19,23): after 9-d wrist immobilization, the wrist flexion decreased by 29% and forearm muscle CSA decreased by 4% (23); after 5-wk arm immobilization, maximal elbow extension strength decreased by 41% and fiber areas of triceps brachii reduced by 25–30% (18,19); after 4-wk unilateral lower-limb suspension, peak torque for the knee flexion decreased by 22% and CSA of the vastus lateralis decreased by 7% (6); and after 17-d space flight, the average peak force dropped by 21% in the soleus slow Type I fiber whereas the CSA decreased by 15% (10). The significant strength loss of 17.9% observed in this current research, in the absence of observed CSA loss, may be caused by change in the nervous system, which may also have altered motor unit recruitment patterns (10). A past study by using functional MRI suggested that relatively untrained people cannot fully activate motor units during a maximal voluntary force of contraction (MVC) even if there is no change in CSA (3). In this study, we measured forearm muscle CSA only at the resting period. For future studies, we must examine whether immobilization will change the forearm muscle recruitment pattern during the maximum grip strength performance from PRE to POST by using functional MRI.
The previous study (12) demonstrated by using 31P-MRS that hand grip contraction exercise at the intensity of 30% MVC and at the frequency of 1 Hz until exhaustion achieved maximal blood flow and maximal oxygen uptake in forearm muscles, and also indicated the full recruitment of the glycolytic system. We selected this protocol as the exercise to fully recruit both oxidative and glycolytic energy systems. After the 21-d immobilization, the average grip repetitions at the absolute load decreased from 49 to 40 times (reduced by 19.1%) for the CAST. One possible explanation of the reduction of grip repetitions at the absolute load is that the grip strength decreased by 17.9% in the postimmobilization, so the relative workload was higher in POST than PRE during the grip endurance. For the absolute load, there was no difference observed between the rate of change of pH and PCr during exercise in PRE and the respective rates of change in POST. The minimum muscle pH reached the same level of 6.5 in both PRE and POST. It is suggested that the dropped muscle pH may be the limiting factor for this exercise, and the source of energy supply from oxidative and glycolytic during the exercise may not change from PRE to POST.
The average grip repetitions at the relative load for the CAST showed no significant change. It is speculated that the glycolytic energy system may fully make up for the oxidative energy loss (the muscle oxidative capacity decreased by 45%). For the relative load, there was no difference found when comparing the intramuscular pH and PCr changes during the grip endurance exercise of PRE and POST. One possible explanation is that the recruitment of energy from the glycolytic energy system during this exercise is much higher than that from the oxidative energy supply. Thus, the large reduction rate of the oxidative capacity has no effect on grip repetition. This hypothesis is supported by the data from a past study. Walter et al. (25) found that during 30-s maximal voluntary planter flexion exercise, ATP provisions from anaerobic ATP sources are much larger than that from aerobic ATP sources.
One notable finding of this research is that the deterioration of forearm muscle function can be produced even if there is no change in muscle morphology. Thus, this research indicates the need to establish an effective countermeasure training program to prevent forearm muscle disfunction even in a short period (up to 21 d) in the microgravity environment.
In conclusion, 21-d forearm immobilization caused no significant changes in forearm muscle morphology but caused remarkable deterioration in muscle function ranging from 18 to 45%, depending on the functions.
The authors are indebted to the six subjects who endured 21-d of forearm immobilization, and we thank Mr. E. Sell for his helpful suggestions in preparing the English manuscript.
This study was funded as part of the Ground Research for Space Utilization program promoted by NASDA and the Japan Space Forum (T. Hamaoka).
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Keywords:©2003The American College of Sports Medicine
MICROGRAVITY ENVIRONMENT; MUSCLE ATROPHY; 31PHOSPHORUS MAGNETIC RESONANCE SPECTROSCOPY; MAGNETIC RESONANCE IMAGING