INTRODUCTION

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THE TECHNIQUE of hindlimb suspension developed by Morey (21) has proved to be a useful animal model for simulation of the effects of weightlessness in a muscular system. Like spaceflight, hindlimb suspension induces significant losses in the hindlimb muscles, with the greatest change observed in antigravity muscles such as the soleus (Sol; 29). The major modifications are muscle atrophy, an increase in fast-twitch fibers, and a decrease in slow-twitch fibers (7, 19).

Rifenberick et al. (24) showed that immobilization reduced mitochondrial enzyme activities, but they did not specify the different subpopulations of mitochondria. Palmer et al. (23) isolated from the heart two different morphological and biochemical populations existing in the same tissue. More recently, several authors (5, 15-17) have confirmed the existence of two populations of mitochondria in the skeletal muscle: the subsarcolemmal (SS) mitochondria, located just beneath the sarcolemma, and the intermyofibrillar (IMF) mitochondria, encased between the myofibrils. These two populations respond differently to muscle contraction or immobilization. For example, Krieger et al. (17) found that 2 days of hindlimb immobilization resulted in a significant 37% decrease in state 3 respiration of the SS mitochondria and no significant change in state 3 respiration of the IMF mitochondria isolated from the gastrocnemius (Gas) muscle. We do not know, however, how a long period of hypodynamia alters mitochondrial respiration. From the literature, we know that 5 wk of hindlimb suspension results in a significant decrease of 26% in the SS mitochondrial volume density in the Sol muscle and a significant increase in the IMF mitochondrial volume density (7). Studies on the biochemical characteristics of SS and IMF mitochondria isolated from skeletal muscle are limited (2, 5, 14, 17) but are potentially important for an understanding of the cellular functions of these mitochondrial fractions in muscle. Recent studies, for example, report that an altered skeletal muscle oxidation capacity may contribute to the exercise-limited capacity in deconditioned patients with heart failure (31) or chronic lung disease (13). Moreover, the hindlimb is constituted of different muscles that are distinguished by their contractile properties and fiber types, i.e., muscles that are either predominantly type I fibers, like the Sol; type II fibers, like the extensor digitorum longus (EDL); or mixed fibers, like the tibial anterior (TA) and Gas.

We hypothesized that long-term reduced muscle activity would differently alter the two mitochondrial subpopulations, as well as the mitochondria isolated from slow- or fast-twitch muscles. To test this hypothesis, we compared the activities of both SS and IMF mitochondria isolated from hindlimb muscles in control rats and 4-wk hindlimb-suspended rats. We also measured mitochondrial respiration and citrate synthase (CS) activity in four single muscles: Sol, EDL, TA, and Gas.

	MATERIALS AND METHODS

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Animal care and muscle extraction. Wistar male rats (260-280 g) were obtained from a local supplier. Rats were divided into two groups of 14 rats each, with animals randomly assigned to either the control or hindlimb-suspension group. They were housed in individual cages, with food and drink ad libitum, in a 12:12-h inverted light-dark cycle. Rats from the suspension group were suspended for 4 wk according to the noninvasive procedure of Morey (21) with modifications (32) used in our laboratory (9). Approximately 5 cm of the distal end of the tail remained uncovered for use as a visual marker of blood circulation throughout the tail. The arrangement allowed the rats to move freely in their cages by using their forelimbs as the only mechanism of movement. They were inspected regularly for safety and for correct angular position.

Reagents. Reagents of the highest quality available were purchased from Sigma Chemical unless otherwise stated.

Enzymatic activities. CS activity was measured in isolated mitochondria according to the method of Srere (27) in a reaction medium consisting of 100 µM dinitrothiobenzoic acid, 100 mM tris(hydroxymethyl)aminomethane (Tris), and 15 µM acetylcoenzyme A. Isolated mitochondria were defrosted at room temperature and submitted to ultrasonication (Sonorex RK 100) for 1 min to expose the CS enzyme, and 10 µg of mitochondrial protein were added to the reaction medium. The absorbance at 412 nm (DU 640 spectrophotometer; Beckman) was checked for 3 min to determine the nonspecific activity caused by to other acetylcoenzyme A deacylases. Reactions were then initiated by addition of 500 µM of oxaloacetate, and the change in absorbance was recorded for at least 3 min at 412 nm. Proteins were estimated by the Coomassie brillant blue method by using bovine -globulin as a standard (Bio-Rad protein assay). Results were expressed in micromoles per minute per gram protein.

Extraction of myofibrils and separation of the MHC isoforms. Separating and stacking gels were prepared according to the detailed procedure of Talmadge and Roy (28) and using the same electrophoresis system (Bio-Rad miniprotean II). A sample of muscle (50-100 mg) was homogenized in 10 volumes of 250 mM sucrose, 20 mM Tris, 5 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA), and 100 mM KCl, pH 6.8, with the use of a glass tissue homogenizer on ice. Homogenates were then centrifuged for 10 min at 4°C at 1,500 g. The myofibril pellets were subsequently washed twice with 175 mM KCl, 20 mM Tris, 5 mM EGTA, and 0.5% Triton X-100, pH 6.8, as described by Tsika et al. (30). Two final washes with 150 mM KCl, 5 mM EDTA, and 20 mM Tris, pH 7.0, gave purified myofibril pellets that were diluted to 3 mg protein/ml. Samples were then diluted at 1 mg/ml in 50 mM pyrophosphate, 50% glycerol, 5 mM EGTA, and 2 mM mercaptoethanol, pH 8.8, and incubated in one volume of denaturing buffer (18) for 5 min at 100°C. Then 3 µg of protein were loaded in each well. The run was at 75 V (constant) for 30 h in a cold room. Overnight staining by Coomassie blue in 40% methanol-10% acetic acid, followed by destaining in 10% methanol-10% acetic acid, revealed several types of MHC isoforms. The gel was scanned (AGFA Studioscan II SI), and the MHC isotypes were quantified with National Institutes of Health software. To do this, we took into account the area and density of each band in the gel. We then calculated the total density of each well; this was normalized to represent 100% so that the MHC subtypes were expressed as a percentage. When we compared each muscle in the control and suspended groups, we found approximately the same total intensity; this result indicates that the same quantity of protein was loaded.

Preparation of the mitochondrial subpopulations. The procedure used for the isolation of the SS and IMF mitochondrial subpopulations was modified from that outlined by Krieger et al. (17) in the medium described by Davies et al. (6). When previously used by our group (12, 20), this medium has proved to be efficient for the isolation of well-coupled mitochondria. Briefly, the hindlimb muscles were removed and immediately placed in ice-cold isolation medium (IM) containing 250 mM mannitol, 10 mM EDTA, 45 mM Tris · HCl, and 5 mM Tris base, pH 7.4. The muscles were freed of fat and connective tissue, transferred into fresh IM, and weighed. All further procedures were carried out at 0-5°C. The muscle sample was next minced with triple scissors in a 1:9 dilution of fresh IM. The muscle sample was homogenized by using a tissue homogenizer (Polytron power control unit) for 10 s with the rheostat set at 5. The effectiveness of the SS extraction from muscles was verified by electronic microscopy after the first polytron treatment (data not shown). The homogenate was centrifuged (Sorvall RC5B Plus) at 800 g for 10 min, and the resulting precipitate was subsequently used for the preparation of the IMF mitochondria. The final SS mitochondrial pellet was suspended in a small amount (2 ml) of IM buffer. The pellet from the 800-g centrifugation contained primarily intact material with some remaining SS mitochondria. This pellet was then washed, and the IMF mitochondria were liberated with a trypsin incubation (0.35 mg/g muscle; 15 min). After two washes were performed, the final IMF mitochondrial pellet was suspended in 1 ml IM. Mitochondria were kept on ice until mitochondrial respiration measurements were made. The final mitochondrial protein concentration was determined by Bradford protein assay (Bio-Rad protein assay) with bovine serum albumin as a standard.

Preparation of the whole muscle mitochondria. The mitochondria were isolated from the four muscles (Sol, EDL, TA, and Gas) according to the method of Davies et al. (6). Briefly, the same IM as described above was used, and, after a trypsin incubation (0.35 mg/g muscle; 30 min), the muscle samples were homogenized. A first centrifugation at 500 g separated the mitochondria. After being washed twice, the final mitochondrial pellets were suspended in 1 ml IM. Mitochondria were kept on ice until mitochondrial respiration measurements were performed.

Mitochondrial respiration. The order of mitochondrial oxygen consumption measurements was random, and all measurements were completed within 2 h of mitochondrial isolation. Rates of oxygen consumption by mitochondria were measured with a complete oxygen-measurement system composed of an oxygen meter (Strathkelvin system, model 781), a microcathode oxygen electrode (Clark type polarographic electrode), and a respiration chamber (3 ml) set at 37°C. When the electrode was assembled, it was calibrated. The zero value was obtained with a sodium borate solution (2.0 ml of a 0.01 M solution) added to the chamber with crystals of sodium sulfite. The maximal value was calibrated with respiration medium alone (1.87 ml) containing 780 × 10⁹ atoms of oxygen. Before measurements of oxygen consumption, the respiration chamber was filled with 1.87 ml of respiratory medium (RM) containing (in mM) 15 KCl, 30 K₂HPO₄, 25 Tris base, 45 sucrose, 12 mannitol, 5 MgCl₂, 7 EDTA, 0.2% bovine serum albumin, and 20 glucose, pH 7.4. One of two substrates (pyruvate + malate or succinate + rotenone) was added to the respiration chamber. Final concentrations were 10 mM pyruvate and 2.5 mM malate or 10 mM succinate and 5.0 µM rotenone to inhibit complex I (NADH reductase) of the electron-transport chain. A capillary-shaped opening in the electrode allowed sequential addition of substrate with no risk of air diffusing into the chamber. Then 1 mg of isolated mitochondria was loaded into the chamber with a magnetic stirring apparatus to ensure complete mixing of mitochondria and substrate. ADP at 250 µM final concentration in a 2-ml chamber final volume was added to initiate state 3 (ADP-stimulated) respiration. The respiratory control ratio (RCR) was obtained by dividing state 3 respiration by the recovery rate after completion of ATP synthesis, i.e., state 4 respiration defined as oxygen consumption of mitochondria after the depletion of exogenous ADP (4). Oxygen consumption was determined as the amount of oxygen disappearing from the respiration chamber over time per 1 mg of mitochondrial protein.

Statistical analysis. Statistical analysis was done by using SigmaStat statistical software. Data are reported as means ± SE. Data concerning the body weights were analyzed by using the Student's t-test, with the 0.05 probability level designated as significant. Data concerning the mitochondrial subpopulations or mitochondria isolated from single muscles were analyzed by a two-way analysis of variance (control vs. hindlimb-suspended groups and SS or IMF mitochondria or mitochondria from each muscle) as previously done (10). The data were obtained from 15 to 20 different oxygen consumption measurements for each substrate in each subgroup of seven animals. The mitochondria were suspended in a final volume of 1.0 ml, and 100 µl of mitochondria were used for each mitochondrial respiration measurement. Thus we could obtain two or three assays for each substrate and for each group. With n = 7 rats, we obtained ~15-20 measurements.

	RESULTS

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Body weight and MHC isoforms after hindlimb suspension. After 4 wk of tail suspension, the animals from the hindlimb-suspended group did not exhibit a significant difference in body weight in comparison with the control group (300-330 g). Figure 1 shows a representative polyacrylamide gel for control and suspended rats. A shift in the soleus muscle after hindlimb suspension from type I (slow) MHC isoforms toward faster types is clearly visible. Moreover, the quantification represented in Table 1 shows a decrease of 38% in type I and an increase of 20 and 18% in types IIx and IIb, respectively. In comparison with the control, the type I MHC of the Gas and the TA was also decreased after suspension.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Polyacrylamide gel of myosin heavy chain isoforms (arrows at I, IIb, IIx, and IIa) of control rats (lanes 1, 3, 5, and 7) and hindlimb-suspended rats (lanes 2, 4, 6, and 8). Lanes 1 and 2, extensor digitorum longus; lanes 3 and 4, soleus; lanes 5 and 6, tibialis anterior; lanes 7 and 8, gastrocnemius.

Protein yields of isolated SS and IMF mitochondria. The protein yields of SS and IMF mitochondria are reported in Table 2. The SS yield decreased 25%, whereas the IMF yield varied by only 5%, but neither of these differences was significant. There was no significant difference between the control and suspended groups in the mitochondrial yield for either subpopulation.

Activities of the SS and IMF mitochondria in the control group. Figure 2, A and B, shows the values of RCR obtained with, respectively, pyruvate + malate and succinate + rotenone as respiratory substrates. The values of RCR indicate that the protocol used for mitochondrial isolation resulted in well-coupled mitochondria. Moreover, the cytochrome-c test confirmed the integrity of the outer membrane, as there was no activation of state 3 after the cytochrome-c was added during state 3.

View larger version (37K): [in this window] [in a new window]   View larger version (40K): [in this window] [in a new window]   Fig. 2.   Respiratory control ratio (RCR) for control and hindlimb suspension rats. This parameter was calculated for subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. Open bars, control group; hatched bars, hindlimb-suspended group. A: values with pyruvate and malate as substrate. B: values with succinate and rotenone as substrate. Data are means ± SE obtained from 7 rats in each group.

View larger version (27K): [in this window] [in a new window]   View larger version (34K): [in this window] [in a new window]   Fig. 3.   O2 consumption of SS and IMF mitochondria during states 3 and 4 for control (open bars) and hindlimb-suspension (hatched bars) groups. A: O2 consumption measurements with pyruvate + malate as substrate. B: O2 consumption measurements with succinate + rotenone as substrate. Values are means ± SE obtained from 7 rats in each group. * Significant difference from control group, P < 0.05; § significant difference between SS and IMF mitochondria from hindlimb-suspension group, P < 0.05.

Effects of hypodynamia on respiration of SS and IMF mitochondria. The SS mitochondria from the control and hindlimb-suspended groups showed similar RCR values for the two substrates considered (Fig. 2, A and B).

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Effects of hindlimb suspension on single muscle wet weights. The results of hindlimb suspension presented in Table 3 show that the Sol, EDL, TA, and Gas muscles had lower wet weights compared with the control group, with decreases of 38, 28, 14, and 32%, respectively (all P < 0.05).

Protein yield and CS activity of mitochondria isolated from single muscles after hindlimb suspension. Table 4 shows the protein yields and CS activity of the mitochondria isolated from muscles (Sol, EDL, TA, and Gas) of control and suspended rats. For all muscles, there was no significant difference in protein yields. The CS activity was significantly lower in the suspended group compared with the control for the EDL, Sol, and TA (P < 0.05). This difference was not significant for the Gas, although it presented a 20% lower value compared with the control group.

Effects of hindlimb suspension on mitochondrial respiration. The results are reported in Table 5. With pyruvate + malate, states 3 and 4 tended to decrease in Sol and TA and to increase in EDL, but there was no significant difference between the control and suspended groups. However, Gas presented a 59 and 42% decrease in states 3 and 4, respectively (P < 0.05).

	DISCUSSION

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We observed that, after a period of 4 wk of hindlimb suspension, the state 3 respiratory rate of the IMF mitochondria was decreased when pyruvate + malate was used as the respiratory substrate. In mitochondria isolated from single muscles, we observed a significant state 3 decrease in the mitochondria isolated from the Gas muscle, whereas the other muscles showed no significant variation. With succinate, no significant difference was noted. The mitochondria from these muscles globally presented a significant decrease in CS activity.

The isolation procedure used in this study allowed us to obtain well-coupled mitochondria, as shown by the high values of RCR and the cytochrome-c test. Also, we confirmed the separation of the SS mitochondria by electronic microscopy, which showed the SS disruption and the empty place of the SS mitochondria. We did not find the same mitochondrial yields obtained by other authors (5, 17, 23), who have reported higher SS yields. We did, however, confirm a higher state 3 respiration in IMF compared with SS mitochondria. The differences in mitochondrial yields could be explained by the type of muscle used in each study. Krieger et al. (17) used the Gas, Cogswell et al. (5) used the quadriceps, and we used the entire hindlimb muscles. However, as in the present study, Barré et al. (2), who used the Gas, found IMF protein yield high compared with the SS protein yield.

We chose the modified tail-suspension model (32) because it is considered less stressful than the harness method. In our experiment, animals from the suspended group showed no significant difference in body weight compared with the control group. Moreover, the stressing effect of suspension was not enough to induce notable changes in the immune system (29), as was shown in an earlier study that measured spleen weight after 4 wk of hindlimb suspension (9). The stress response of the animal to hindlimb unweighting is transient and minimal in magnitude (29). We concluded that the stress effects that occurred after 4 wk of hindlimb suspension were minimal and that the further modifications observed were caused more by the reduction of activity. To verify efficiency of suspension, we studied the MHC isoform distribution, a parameter known to vary during hindlimb suspension (29). Our electrophoretic pattern of purified myofibrils from the Sol muscle showed a decrease in the type I slow MHC isoform and an increase in faster MHC isoforms (8, 9).

We found a higher IMF mitochondrial respiratory rate for control rats in comparison with the SS fraction. This is in agreement with other findings (2, 5, 17, 23). After hypodynamia, this difference remained. Moreover, this result confirmed the effective extraction of the two subpopulations from the skeletal muscle. Mitochondrial yield was not significantly different between the control and hindlimb-suspension groups, indicating that 4 wk of hypodynamia did not cause a decline in mitochondrial content. These results are in agreement with the study of Joffe et al. (14), who reported that SS and IMF mitochondrial yield was not significantly decreased after 21 days of inactivity induced by denervation. We can speculate that the SS mitochondria decreased quantitatively, whereas the IMF mitochondria decreased more qualitatively. Indeed, the yield decreased more for the SS mitochondria (25%) than for the IMF mitochondria (6%). Although the decrease in SS mitochondria was not significant, it clearly indicates a quantitative change. This result is in agreement with Riley et al. (25), who reported the SS mitochondria degraded more rapidly than the IMF mitochondria after hindlimb suspension.

We suggest that the IMF mitochondria decreased qualitatively because, with pyruvate + malate as respiratory substrate, state 3 in these mitochondria decreased significantly. Because we observed a decrease in mitochondrial oxygen consumption with pyruvate + malate but not with succinate + rotenone as substrate after 4 wk of hindlimb suspension, our results indicate that hypodynamia may act negatively on complex I or prior sites, but not on complex II of the electron transport chain.

The literature reports conflicting results concerning the effect of hindlimb hypodynamia on skeletal muscle mitochondrial activity. Krieger et al. (17) showed that hindlimb immobilization resulted in decreased activity of complex I of the respiratory chain in SS mitochondria without any significant decrease in IMF activity. Joffe et al. (14) observed a decrease, after denervation, in both SS and IMF mitochondrial activity. These differences can be explained by the different durations of muscle hypodynamia and the methods used to induce it. Indeed, in the study by Krieger et al. (17), the duration of immobilization was only 2 days, and the immobilization was achieved with plaster of paris. We can speculate that the hindlimb was immobilized in such a condition, but that the muscle was still able to contract against the plaster. This may explain why these authors did not observe a decrease in IMF mitochondria, which are involved in energy production necessary for muscle contraction (3). Moreover, in contrast to the model of disuse used by Krieger et al., the suspension technique does not restrict the hindlimb muscles from a full range of voluntary isotonic contractions. The present study showed that IMF mitochondria are more sensitive to hindlimb suspension. One plausible explanation is that the apparent hindlimb-suspension-mediated modification in muscle fiber type is dependent on the elimination of load bearing or isometric contraction. In the study done by Joffe et al. (14), the hypodynamia was longer and more comparable to the duration of the hindlimb suspension in the present study. However, their differences compared with the results of the present study can be explained by other effects of denervation on skeletal muscle, such as modification in vascularization and decrease in mitochondrial activity (9a).

Another objective of the present study was to determine which type of muscle is most affected by hindlimb suspension. After 4 wk of hindlimb suspension, Sol, EDL, TA, and Gas muscles all presented significant decreases in wet weight, indicating a dramatic muscle atrophy. We found no significant variation in the mitochondrial protein yield. This result is in agreement with the fact that hypodynamia did not cause a loss of overall mitochondrial content (14). We can thus conclude that skeletal muscle atrophy is not concomitant with mitochondrial protein loss. When we investigated oxidative metabolism, we noted a significant decrease in CS activity of the Sol, EDL, and TA compared with the control; the Gas also presented decreased activity, although the difference did not reach significance. After 4 wk of hindlimb suspension, Gas mitochondria presented the greatest decrease in mitochondrial respiration. This muscle seems to be very sensitive to the degree of muscle inactivity; Krieger et al. (17) showed a decrease in Gas mitochondrial respiration after 2 days of immobilization. In the present study, we found a decrease in the type I MHC. Although the decreased CS activity was not significant, this enzyme activity was 20% lower compared with the control group. Moreover, the literature reports muscular atrophy that is not accompanied by a reduction in the CS activity. For example, Thomason and Booth (29) reported an atrophy induced by 28 days of unweighting, and CS activity was not modified. Musacchia et al. (22) found that the area of type I and II fibers area was significantly reduced in rats after 14 days of spaceflight, and the CS activity showed no differences in the flight group compared with the control group.

Baldwin et al. (1) showed that, compared with the control group, pyruvate oxidation capacity and the marker of oxidative enzymes were not altered in rats after 9 days of spaceflight. These authors also observed that spaceflight induced a decline in palmitate oxidation. These results are comparable to those that we obtained with EDL, Sol, and TA. Indeed, we observed no modification in the respiration of mitochondria with pyruvate + malate and a reduction in an oxidative marker, the CS activity.

In summary, we observed that 4 wk of hindlimb suspension altered IMF mitochondria more than SS mitochondria and seemed to act negatively on complex I of the electron transport chain or prior sites. The mitochondria most affected were those isolated from the Gas muscle.