Skeletal muscle is susceptible to morphological and functional changes when exposed to altered (increased or decreased) mechanical load. The impact of unloading (e.g., weightlessness or hindlimb suspension) on skeletal muscle is more pronounced in slow-twitch (e.g., soleus) antigravity muscles than in fast-twitch muscle (e.g., extensor digitorum longus (3,5,15,22). Unloading of antigravity muscles leads to muscle atrophy and conversion of biochemical and functional properties resembling those more typical of fast-twitch fibers (3,5,15,22,24). In contrast, mechanical overload of skeletal muscle results in muscle hypertrophy (10,19,24) and conversion of functional and structural properties resembling those typical of slow-twitch skeletal muscle (19,24).
Reloading (i.e., return to 1 gravity or weight bearing) of skeletal muscle after a period of unloading is in many respects similar to mechanical overload. This process (i.e., reloading) also has a profound effect on muscle morphology and ultrastructure. Morphological changes occur within 5 h of reloading and include an increase in muscle wet weight and cross-sectional area (17), swelling of the fibers leading to a more rounded contour (13), and appearance of eccentric contraction-like sarcomere lesions (17). The incidence of sarcomere lesions increases from 5 to 48 h post reloading (17). Although these studies indicate that muscle injury occurs as a consequence of reloading, the impact of reloading after short-term hindlimb suspension on contractile function is not clear.
The occurrence of damage in unloaded muscles may have important functional consequences. Because most studies of muscle repair have been performed in a normal 1-gravity environment, it is unclear to what extent removal of load will have on the repair process and functional recovery. Some studies indicate that absence of load (e.g., hindlimb suspension) can significantly impair the rate of muscle regeneration (7), whereas increased load (via exercise) can significantly increase recovery rate (11,12). However, none of these studies directly measured recovery of contractile function. Therefore, the purposes of this study included: 1) to determine the impact of reloading after 7-d hindlimb suspension in the rat on the in vitro isometric contractile properties of the soleus muscle, and 2) to determine the impact of altered mechanical load (i.e., unloading, exercise) on the recovery of muscle contractile properties after reloading-induced injury.
Two experiments were performed. First, the impact of reloading after 7 d of hindlimb suspension on the in vitro isometric contractile properties of the soleus muscle was determined (experiment 1). In the second study, the impact of altered mechanical load on recovery of muscle contractile properties after reloading-induced muscle injury was examined (experiment 2).
Young adult male Sprague-Dawley rats (240–270 g) were housed in the Laboratory Animal Facility. All animals were provided with commercial rat chow and water ad libitum and maintained on a 12-h light–dark cycle. All procedures were approved by the University at Buffalo Institutional Animal Care and Use Committee.
In experiment 1, all rats were hindlimb suspended for a period of 7 d and studied immediately afterward (no reloading, 0D-RL, N = 5), after 1 d (24 h) of reloading (1D-RL, N = 7), or after 2 d (48 h) reloading (2D-RL, N = 8). After each of these time points, in vitro isometric contractile properties were measured in the soleus muscle.
In experiment 2, all animals underwent hindlimb suspension for 7 d followed by reloading for 2 d (48 h). After the 2-d reload period, rats were randomly assigned to one of three groups: 1) cage-bound, 2) treadmill walking, or 3) re-hindlimb suspension (resuspended). The rats in the cage-bound group were allowed to ambulate within their cages, whereas the treadmill walking group (in addition to being cage-bound) participated in forced daily treadmill walking (12 m·min-1, 0% grade, 20 min·d-1). Rats from each group were studied either 7 or 14 d after the 2-d reload period. For the 7-d recovery period, sample sizes were 8, 8, and 7 for cage-bound, treadmill walking, and resuspension groups, respectively, whereas for the 14-d recovery period sample sizes were 8, 8, and 6 for the same respective groups.
Hindlimb suspension procedure
Hindlimb suspension was achieved using a tail harness. Before harness attachment, the rat was placed in a restraining device with the tail exposed. The tail harness was made from a rectangular piece (4 cm × 1.5 cm) of plastic tubing padded with a vinyl strip. A piece of wire was attached perpendicular to both sides of the tail harness. The tail was cleaned with 70% alcohol, and rubber cement was then applied to both the harness and proximal one-third of the tail. Once the rubber cement dried, the tail was firmly placed on the harness. The tail and harness together were then wrapped with orthopedic and elastoplastic tape. The harness was then attached, via a metal wire, to a swivel connector suspended above the cage such that the rat was in a ∼45° head down angle. The swivel was connected to a wire that ran along the top of the cage. Movement of the swivel along the wire was restricted such that the rat at no time could push off the sides of the cage with its back feet. The forelimbs maintained contact with the floor of the cage such that the animals were able to move about to obtain food and water.
Measurement of muscle contractile properties
The animals were anesthetized with a mixture of ketamine (60–80 mg·kg-1) and xylazine (2–10 mg·kg-1) injected intraperitoneally. Once a surgical plane of anesthesia was reached, the soleus muscles were rapidly excised from both limbs and placed in cooled (4°C) Krebs solution (137 mmol NaCl, 4 mmol KCl, 1 mmol MgCl2, 1 mmol KH2PO4, 12 mmol NaHCO3, 2 mmol CaCL2, 6.5 mmol C6H12O6) aerated with 95%O2 and 5% CO2. One end (origin) of the soleus tendon was attached to a stiff polycarbonate strip 1 mm in width, which in turn was secured to the lever arm of the Dual Channel Force-Length Servo Control System (Cambridge Technologies, Model 305B, Andover, MA). The other end (insertion) of the soleus tendon was clamped to a metal rod, which in turn was fixed to a micropositioner. The soleus muscle was placed in a glass tissue chamber perfused with aerated Krebs solution maintained at 25°C.
The soleus muscle was stimulated using large platinum electrodes (1 cm × 4 cm) using monophasic rectangular pulses (0.2 ms in duration) of anodal current (Grass model S48 stimulator with current amplifier, Grass Instrument, a division of Astro-Med, West Warwick, RI). Stimulus intensity was increased until peak twitch force (Pt) was reached. Muscle length was then incrementally increased until optimal muscle length (Lo) was found for Pt. Lo was measured with a micrometer to the nearest tenth of a mm. After determination and measurement of Lo, a force-frequency curve was obtained using stimuli in 500 ms trains at frequencies ranging from 10 to 100 Hz in order to obtain maximal isometric force (Po). After the completion of the contractile studies, the muscle was removed from the chamber and weighed to the nearest mg. CSA of the muscle was determined according to the following formula:MATH
where MW = mass of the muscle in grams, Lf = optimal fiber length in cm, and 1.056 = the density (g·cm-3) of the muscle (16). Lf was estimated to be 2/3 of Lo based on results from previous studies (4,14,20). All forces were normalized for muscle CSA and expressed as N·cm-2.
Mean values of the dependent variables for the three groups in experiment 1 were compared using a one-way analysis of variance (ANOVA) and post hoc analysis (Bonferroni). In experiment 2, a repeated measures two-way ANOVA was used to assess the effect of time and group on body weight. For other measures, a two-way ANOVA statistical design was used to assess the main effects (load and time). A Bonferroni post hoc analysis was used where the F-value indicated overall significance (P < 0.05).
The body weights for rats studied ranged from 250 to 270 g. The mean soleus wet weights are illustrated in Figure 1. Compared with the 0D-RL group, soleus muscle wet weight was significantly increased in both the 1D-RL and 2D-RL groups.
Specific Po (N·cm-2) was significantly decreased (P < 0.05) by ∼32% and ∼50% in the 1D-RL and 2D-RL groups, respectively, when compared with the 0D-RL group (Fig. 2). To eliminate the potential confounding influence of interstitial edema, maximal force generation is also plotted in newtons (N) (Fig. 3). When expressed in N, Po was ∼21% and 33% (P < 0.05) lower in the 1D-RL and 2D-RL groups, respectively, when compared with the 0D-RL group.
The mean body weights for all groups in experiment 2, recorded before hindlimb suspension, after 2 d reloading, and at 7 or 14 d recovery (i.e., after the 2-d reload period), are illustrated in Figures 4 and 5, respectively. The body weights did not significantly change from before hindlimb suspension to after the 2-d reloading period. After 7 d of recovery, body weights significantly (P < 0.05) increased in the cage-bound and treadmill groups but not in the resuspended group (Fig. 4). After 14 d of recovery, body weight significantly increased in all groups (P < 0.05) but to a greater extent in the cage-bound and treadmill groups (Fig. 5).
The soleus muscle wet weights in experiment 2 are illustrated in Fig. 6. Seven and 14 d into recovery, soleus muscle wet weight was significantly higher (P < 0.05) in the cage-bound and treadmill groups compared with the resuspension group (Fig. 6). However, for a given group, muscle weight did not significantly change between 7 and 14 d.
Soleus muscle Lf and CSA for experiment 2 are shown in Table 1. Seven days into recovery, Lf and CSA were significantly (P < 0.05) less in the resuspension group compared with the cage-bound and treadmill walking groups. However, after 14 d of recovery, only muscle CSA of the resuspension group was significantly (P < 0.05) lower than the cage-bound and treadmill walking groups.
Maximal isometric force (N) of the soleus muscle at 7- and 14-d recovery is illustrated in Figure 7. Relative to the cage-bound and treadmill groups, soleus muscle maximal isometric force was significantly less (P < 0.05) in the resuspension group for each time point. For a given group, there was no significant change in maximal isometric force from 7 to 14 d recovery.
After 7 d recovery, specific Po was significantly lower (P < 0.05) in the resuspension group compared with cage-bound and treadmill walking groups, whereas the latter two groups did not differ from one another (Fig. 8). Fourteen days into recovery, a similar trend was observed. Although specific Po increased slightly in the cage-bound and treadmill walking groups (compared with 7 d), the increase was not significant. Additionally, there was no change in specific Po in the resuspension group from 7 to 14 d recovery.
In the present study, reloading of the soleus muscle for 1 or 2 d after 7 d of hindlimb suspension resulted in a significant decrease in Po, the latter time point resulting in a greater decrease. It was also observed that removal of load, i.e., resuspension, significantly impaired recovery of in vitro isometric contractile properties, whereas forced treadmill walking over a 2-wk period had no added benefit over normal cage-bound activity.
Compared with 7 d hindlimb suspension, soleus muscle mass was significantly increased after 1- and 2-d reloading. Previous studies examining the impact of acute remobilization (13) or acute compensatory overload (10) have found a significant increase in interstitial space, indicative of muscle edema. Given that the muscle wet weight in this study did not significantly change from 1- to 2-d reload, any change in interstitial space presumably occurred within the first 24 h reload. Although interstitial space was not measured in this study, the observed acute change in wet weight after reloading (a process similar to compensatory overload) is consistent with previous studies (10,13,17).
Reloading also significantly affected muscle contractility. Specific Po significantly decreased ∼32% and ∼50% 1 and 2 d after reloading, respectively. Specific Po (i.e., force/CSA) can be affected by alterations in the integrity of the myofibrillar architecture (e.g., muscle injury) and by factors that influence CSA, e.g., edema. When Po was expressed in newtons to eliminate the potential confounding variable of increased water content, Po was still decreased both 1 and 2 d after reloading, albeit to a smaller extent. Kandarian and White (10) determined that during acute compensatory overload, muscle edema accounted for ∼50% of the observed decrease in Po. The decrease in Po in this study was ∼50% less when expressed as N rather than as N·cm-2 and is consistent with that previously reported (10). Thus, although the force deficit is less expressed in absolute terms than when normalized for CSA, a significant force deficit still exists 2 d after reload. The decrease in Po, especially when expressed in absolute units, is likely a result of muscle injury, and is consistent with the changes in muscle ultrastructure reported by Krippendorf et al. (13,17). It should be noted however that other factors (e.g., failure of excitation-contraction coupling) might also contribute to the decline in Po.
All rats displayed an initial loss of body weight during the 7-d hindlimb suspension and the 2-d reloading period. Previous studies have shown that acute hindlimb suspension induces a stress response thereby resulting in an elevated plasma glucocorticoid concentration (2,9,21). Armstrong et al. (2) found that mice subjected to 11 d hindlimb suspension lost ∼8.6% in body weight and had serum corticosteroid levels that were approximately double that observed in control animals. However, Steffen and Musacchia (21) found plasma corticosterone concentrations in rats to be significantly elevated only during the first 3 d of hindlimb suspension with a return to normal levels by the 7th day. The return to basal glucocorticoid levels observed by Steffen and Musacchia (21) may in part explain why all the rats in our study subsequently gained weight during the resuspension period, albeit to a smaller extent during the first 7 d.
The amount of mechanical load the soleus muscle was subsequently exposed to during the recovery phase significantly affected soleus muscle mass. By 7 d recovery, the wet weight of the soleus muscle in the resuspended group was similar to that observed in the 0D-RL group from experiment 1. There was no further change in soleus wet weight from 7 to 14 d recovery with resuspension, even though body weight significantly increased. In contrast, the wet weight of the soleus muscle increased in both the cage-bound and treadmill walking groups 7 and 14 d into recovery. The mechanism responsible for the marked differences in muscle mass is not clear. The differences in muscle weight as a consequence of resuspension versus cage-bound activity or treadmill walking may be the result of depressed satellite cell mitotic activity as a consequence of resuspension (6). Reduced satellite cell mitotic activity may result in the incorporation of fewer satellite cell nuclei into the myofibers. It has been suggested that a reduced quantity of DNA may prevent muscle hypertrophy (1). Future studies are required to determine whether resuspension suppresses satellite cell mitotic activity after reloading-induced damage.
During the recovery phase, Po increased slightly from the 2-d reload period to 7 d resuspension, but there was very little increase in Po from 7 to 14 d resuspension. In contrast, Po increased to a greater extent in the cage-bound and treadmill walking groups compared with resuspension. There was a small nonsignificant increase in Po from day 7 to day 14 in both cage-bound and treadmill walking groups, with Po averaging ∼17.5 and 18.5 N·cm-2, respectively. Thus, although Po approached normal values recorded in our laboratory (∼20 N·cm-2), 2 wk time was insufficient to obtain full functional recovery. These findings are similar to other studies in which achieving complete functional recovery after remobilization (5) or inducement of muscle injury (4) may require 28 d or greater.
It is unclear why Po failed to increase during the 2nd week of resuspension. One possibility is that blood flow to the muscle is decreased during tail suspension (15). The decreased blood flow to the muscle may hamper the muscle’s ability to repair itself because adequate blood flow is necessary for the repair process. Another possibility is that the expression of various cytokines and growth factors involved in the repair process are decreased in the injured unloaded muscle, even during repair. It is known that growth hormone (GH) secretion by the pituitary gland is decreased after hindlimb suspension (25). However, the impact of reduced circulating levels of GH on the repair process during tail suspension is unknown and warrants further study. Lastly, it has been reported that Po significantly decreases as a consequence of prolonged hindlimb suspension, suggesting that the contribution of myofibrillar protein to total CSA decreases (8). It is also possible that recovery of Po during resuspension is at least partially confounded by changes in the myofibrillar to nonmyofibrillar protein ratio (23).
Although resuspension significantly affected the degree of functional recovery, 20 min of daily TM walking was no more beneficial than cage-bound activity alone. The results of this study contrast somewhat to other published studies. For example, Kasper et al. (12) found greater incidence of damaged soleus muscle fibers in exercised-trained rats as compared to cage-bound rats 7 d after remobilization. Rats in this latter study were hindlimb suspended for 28 d before remobilization in contrast to 7 d used in the present study. Additionally, rats were exercised on a treadmill at a speed of 22m·min-1 compared with 12 m·min-1 used in this study. Thus, it maybe that the combination of a longer period of hindlimb suspension before reloading coupled with a higher exercise intensity used during the recovery period resulted in greater fiber damage. Interestingly, although there was a higher incidence of damaged fibers in the study by Kasper et al. (12), the higher exercise intensity resulted in greater fiber size and muscle CSA compared with cage-bound rats. Kannus et al. (11) reported that both mild (12 m·min-1) and moderate (18 m·min-1) treadmill exercise for 8 wk promoted recovery of muscle mass after 3-wk hindlimb suspension. However, other studies have shown that acute muscle growth is not coupled with increases in Po (10). Given that contractile properties were not measured in these studies, it is not clear if exercise facilitated the rate of functional recovery. Our data suggests that mild exercise had no added benefit over cage-bound activity, at least up to 2 wk post-reloading.
In conclusion, acute reloading of the soleus muscle after short-term hindlimb suspension causes a significant decrease in Po, a finding consistent with the morphological and histological changes indicative of muscle damage previously observed (13,17,18). In addition, recovery from reloading-induced injury appears to be significantly impaired when load is removed. Results from this study also showed that treadmill walking in comparison with cage-bound activity did not accelerate the rate of functional recovery as a consequence of reloading induced damage. These findings suggest that mechanical load is an important factor during the repair process, but the amount needed for optimal recovery remains undetermined.
The authors are grateful to Drs. Gaspar Farkas and Michael Hudecki for their critical review during the preparation of this manuscript. This work was supported by the University at Buffalo Moir B. Tanner and Mark Diamond Research Funds.
Address for correspondence: Luc E. Gosselin, Ph.D., Department of Physical Therapy, Exercise and Nutrition Sciences, 405 Kimball Tower, SUNY @ Buffalo, Buffalo, NY 14214; E-mail: [email protected]
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