The FASEB Journal express article 10.1096/fj.00-0629fje. Published online March 12, 2001.

Space shuttle flight (STS-90) enhances degradation of rat myosin heavy chain in association with activation of ubiquitin-proteasome pathway Madoka Ikemoto*, Takeshi Nikawa*, Shin’ichi Takeda†, Chiho Watanabe‡, Takako Kitano*, Kenneth M. Baldwin‡, Ryutaro Izumi§, Ikuya Nonaka�, Takae Towatari#, Shigetada Teshima*, Kazuhito Rokutan*, and Kyoichi Kishi* *Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima 770-8503, Japan; †National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo 187-8502, Japan; ‡Department of Physiology and Biophysics, University of California, Irvine, California 92697, USA, §National Space Development Agency of Japan, Tokyo 105-0013, Japan; �Department of Neuromuscular Research, National Center of Neurology and Psychiatry, Tokyo 187-8551, Japan, #Division of Enzyme Chemistry, Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan Corresponding author: Takeshi Nikawa, M.D., Department of Nutrition, School of Medicine, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. E-mail: nikawa@nutr.med.tokushima-u.ac.jp ABSTRACT To elucidate the mechanisms of microgravity-induced muscle atrophy, we focused on fast-type myosin heavy chain (MHC) degradation and expression of proteases in atrophied gastrocnemius muscles of neonatal rats exposed to 16-d spaceflight (STS-90). The spaceflight stimulated ubiquitination of proteins, including a MHC molecule, and accumulation of MHC degradation fragments in the muscles. Semiquantitative reverse transcriptase-polymerase chain reaction revealed that the spaceflight significantly increased mRNA levels of cathepsin L, proteasome components (RC2 and RC9), polyubiquitin, and ubiquitin-conjugating enzyme in the muscles, compared with those of ground control rats. The levels of µ-calpain, m-calpain, cathepsin B, and cathepsin H mRNAs were not changed by the spaceflight. We also found that tail-suspension of rats for 10 d or longer caused the ubiquitination and degradation of MHC in gastrocnemius muscle, as was observed in the spaceflight rats. In the muscle of suspended rats, these changes were closely associated with activation of proteasome and up-regulation of expression of mRNA for the proteasome components and polyubiquitin. Administration of a cysteine protease inhibitor, E-64, to the suspended rats did not prevent the MHC degradation. Our results suggest that spaceflight induces the degradation of muscle contractile proteins, including MHC, possibly through a ubiquitin-dependent proteolytic pathway. Key words: tail-suspension • proteasome • cathepsin L • ubiquitination

keletal muscles, especially antigravity slow-twitch muscles, are vulnerable to rapid and marked atrophy under microgravity conditions. Studies using non-weight-bearing animals have provided evidence that increased protein breakdown is, at least in part, responsible for

the muscle wasting, in addition to the decline of muscle protein synthesis (1–3). There are three major proteolytic pathways in muscle protein catabolism: a Ca2+-dependent, lysosomal and an ATP-ubiquitin-dependent proteolytic pathway. The activities of lysosomal or cytosol cysteine proteases increase with atrophy of unweighted muscles (1). In addition, a Ca2+ antagonist suppressed the accelerated proteolysis caused by unweighting and a lysosomotropic agent, chloroquine, diminished the atrophy of denervated muscle (3). However, Taillandier et al. have reported that tail-suspension increased the mRNA levels of ubiquitin, a 14-kDa ubiquitin-conjugating enzyme (E214K) and the RC2 and RC9 subunits of 20S proteasome in the atrophied muscles (4). Thus, it is still not clear which proteolytic pathways predominantly mediate the degradation of muscle proteins under unweighting conditions. Myosin heavy chain (MHC) is a major structural protein that controls the intrinsic contractile properties (force generation and shortening) of a muscle fiber (5). This protein is sensitive to various proteases in the major proteolytic pathways, such as proteasome and cathepsins B and L in vitro (6–9). Recently, it was reported that subtle alterations in the rate of either anabolic or catabolic metabolism of MHC can markedly change the cardiac muscle mass (10), which suggests that MHC may be a useful molecule to assess the catabolic state of striated muscles. In the present study, we focused on the degradation of fast-type MHC in atrophied gastrocnemius muscle of neonatal rats exposed to spaceflight (STS-90). The spaceflight stimulated the ubiquitination of muscle proteins, including MHC, and caused the accumulation of MHC degradation fragments. The spaceflight also significantly increased the mRNA levels of 20S proteasome components (RC2 and RC9) and polyubiquitin, which suggests that up-regulation of the ubiquitin-mediated proteolytic pathway may be associated with muscle protein degradation under microgravity conditions. MATERIALS AND METHODS Spaceflight rats As described in detail in a previous report (11), timed-pregnant female rats were obtained from Taconic Farms (Germantown, N.Y.) and housed initially in standard rodent cages in the vivarium at Kennedy Space Center in Florida. Shortly after birth, each litter was adjusted to 6 pups and matched for gender distribution. A cohort of those litters was randomly assigned to the following experimental groups: asynchronous ground control, vivarium ground control, and flight-based groups. In this mission (STS-90), the flight rats were launched at 13:19 on April 17, 1998, into space with the space shuttle Columbia, when they were 8-(P8) or 14-d old (P14). Shuttle rats in P8 and P14 groups were housed with dams in research animal holding facility (RAHF) and animal enclosure module (AEM) cages, respectively, in the orbiter. The RAHF cage was modified from the AEM cage to additionally provide a “nursing” area. During spaceflight, the newborn rats suckled from their dams in addition to eating food bars. The shuttle landed at Kennedy Space Center at 10:10 on May 3, 1998. Approximately 2 h elapsed under weight-bearing conditions before the animals were killed, and isolation of gastrocnemius

S

muscles from all animals was completed within 75 min. Isolated gastrocnemius muscles were weighed and immediately frozen in chilled isopentan and liquid nitrogen. The asynchronous ground control rats were housed with dams in cage conditions that simulated the shuttle’s environment, including the shuttle’s ambient temperature (23–28°C), the facilities, and the timing of events of the flight animals. The vivarium ground control rats were housed in a standard stainless cage in a room maintained at 23°C during the same period of spaceflight. As reported previously (11, 12), rats from these two ground-based control groups had similar values for the measured variables, such as body weight, muscle weight, and protein and mRNA levels of MHC isoforms in skeletal muscle. To simplify the presentation of results, the rats in vivarium cages are referred to here as the ground control group. Tail-suspended rats Male Wistar rats (Japan SLC, Shizuoka, Japan), 5 wk old, were housed in a room maintained at 23°C on a 12-h light/dark cycle and were allowed free access to a 20% casein diet and water. When their body weights reached 200 g (day 0), a group of randomly selected rats were subjected to tail-suspension hypokinesia for the indicated periods by using an apparatus originally described by Morey with minor modifications (13). Their tails were suspended to keep their rear legs off the ground. Food intake of tail-suspended rats decreased to about 80% of that of nonsuspended rats. Pair-fed control rats without suspension were prepared for the same duration. In separate experiments, 2 or 4 mg/rat of trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64), a kind gift from Taisho Pharmaceutical Co. (Tokyo, Japan), was subcutaneously injected into tail-suspended rats twice a day for 2 wk. Phosphate-buffered saline (PBS) as a vehicle was injected into suspended or nonsuspended rats for the same period. All of the treatments described here were performed according to the Guide for the Care and Use of Laboratory Animals (1985) and were approved by the Animal Care Committee of National Aeronautics and Space Administration (NASA) or National Space Development Agency of Japan (NASDA) counterpart. Sample preparations Frozen gastrocnemius muscle was pulverized and then homogenized for 1 min on ice with a Polytron homogenizer in three volumes of ice-cold 20 mM Tris-HCl buffer, pH 7.5, containing 5 mM EDTA and 10 mM dithiothreitol. After separating unbroken tissue, the homogenate was centrifuged at 30,000 x g for 30 min at 4°C and the supernatant was stocked as a soluble fraction for the measurement of calpain or proteasome activity. The pellet was further extracted with the same volume of 50 mM sodium acetate buffer, pH 5.0, containing 0.2 M NaCl and 0.1% Triton X-100 and centrifuged at 13,000 x g for 30 min at 4°C. The supernatant (extract fraction) was used for the measurement of lysosomal cysteine protease activities. To prevent degradation during the preparation, samples for Western blot analysis were prepared by using the Tris- or acetate-based buffer containing a protease inhibitor cocktail set (Roche Diagnostics, Tokyo, Japan). Total RNA was isolated from the tissue with an acid guanidinium thiocyanate-phenol-chloroform mixture (Nippon Gene, Toyama, Japan).

Measurement of muscle protease activity After separating calpastatin from the soluble fraction by phenyl-sepharose column chromatography, calpain activity was determined by measuring hydrolysis of succinyl-Leu-Leu-Val-Tyr-7-(4-methyl)-coumarylamide as described previously (14). Activity of cathepsins B+L was measured in the extract fraction with benzyloxycarbonyl-Phe-Arg-7-(4-methyl)-coumarylamide, as previously described (15). Proteasome activity in the soluble fraction was measured as succinyl-Leu-Leu-Val-Tyr-7-(4-methyl)-coumarylamide-hydrolyzing activity in the presence of 0.05% sodium dodecyl sulfate (SDS), according to the method of Ugai et al. (16). Protein concentrations were measured by Lowry’s method by using bovine serum albumin as a standard. Western blot analyses Myosin degradation fragments were detected by Western blot analysis according to the method of Ball et al. with a minor modification (17). Proteins (40 µg/lane) in the soluble fraction were subjected to SDS/6%-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene difluoride membrane at 35 mA for 6 h at 4°C. The membrane was blocked with 3% skim milk and then incubated for 1 h at 25°C in PBS with a 1:500 dilution of monoclonal antiserum against rabbit skeletal fast-type MHC (Sigma, St. Louis, Mo.). Bound antibodies were detected by using the enhanced chemiluminescence system (Amersham, Little Chalfont, England). Ubiquitinated proteins were detected in the same manner by using a monoclonal antibody against rabbit ubiquitin (Sigma). Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) To measure the expression of protease transcripts, semiquantitative RT-PCR was performed according to the method of Mathur et al. (18). Briefly, first-strand cDNAs were reverse-transcribed at 42°C for 60 min from 1 µg of total RNA with oligo-dT15 primer and avian myoblastosis virus RT (Promega, Madison, Wis.). Following initial denaturation for 5 min at 94°C, second-strand synthesis and DNA amplification with Taq polymerase (Promega) were accomplished through 25–35 cycles of the following incubations: 1 min at 94°C, 2 min at 58°C, and 3 min at 72°C by using a thermal cycler (MJ Research Inc., Watertown, Mass.). PCR buffer contained two sets of primers to simultaneously amplify a target gene cDNA and an internal standard β-actin cDNA. In separate experiments, we performed a DNA microarray analysis on these space samples and found that the level of β-actin mRNA was not changed by the spaceflight (data not shown). The forward and reverse primers used in this study were summarized in Table 1. The amplification was terminated 15 min later at 72°C when PCR products were linearly amplified. The PCR products were separated by electrophoresis in an 8% polyacrylamide gel and detected with a highly sensitive nucleic acid staining reagent (Takara, Tokyo, Japan). The intensities of staining of the target bands, and of internal standard gene cDNAs, were estimated with an image analyzer (FMBIO II, Takara), and the intensity ratio of a target gene cDNA to the internal standard gene cDNA was calculated. Statistical analysis

All data are expressed as mean ± SD and were statistically evaluated by analysis of variance (ANOVA) with SPSS software (release 6.1; SPSS Japan Inc., Tokyo, Japan). One-way ANOVA was used to determine the significant effects of spaceflight, tail-suspension, and administration of E-64 on the variables measured. Individual differences between groups were assessed using Duncan’s multiple range test. Differences were considered significant at P < 0.05. RESULTS Muscle weight The spaceflight significantly decreased the actual wet weights of gastrocnemius muscles in both groups (Fig. 1A). In the P8 group of spaceflight rats, the muscle weight standardized to body weight (mg/g body) decreased, which suggests that younger rats may be more sensitive to muscle atrophy caused by spaceflight. The tail-suspension also decreased in the standardized sizes of hindlimb muscles, compared with those of pair-fed control rats (Fig. 1B). Among the hindlimb muscles, the soleus muscle exhibited the most rapid and severe atrophy. MHC degradation in gastrocnemius muscle under microgravity conditions In both the P8 and P14 spaceflight rats, the levels of a fast-type 200-kDa MHC protein in gastrocnemius muscle decreased, compared with those in the respective ground control rats (Fig. 2A). Concomitantly, the levels of MHC degradation fragments with a molecular mass of approximately 180, 160, 145, 140, 130, or 120 kDa increased in the muscle of spaceflight rats. These proteins were confirmed to be the degradation products of MHC, because incubation of purified MHC with papain produced fragments of the same molecular sizes (data not shown). In addition, the molecular sizes of MHC degradation fragments reported here were similar to those of chicken skeletal MHC degradation fragments reported by Ball et al. (17). The immunoreactive proteins corresponding to the MHC degradation products had significantly accumulated by day 10 and continued to increase in tail-suspended rats, but not in the control rats (Fig. 2B). The severest degradation of MHC was observed on the last day of tail-suspension (day 21). Expression profile of muscle proteases Semiquantitative RT-PCR revealed that the exposure of rats to spaceflight greatly modified the mRNA levels of muscle proteases. The mRNA levels of components (RC2 and RC9) of 20S proteasome, E214K, and polyubiquitin were up-regulated by the spaceflight compared with the respective ground control rats (Fig. 3A). The RT-PCR technique used in this study could amplify the two transcripts of the ubiquitin genes; a monomeric form and a polyubiquitin form (encoding repeated ubiquitin moieties in tandem) (19). Spaceflight increased the levels of polyubiquitin mRNAs encoding 2-, 3-, 4-, 5-, and 7-repeated ubiquitin moieties in tandem (indicated as open arrowheads in Fig. 3A), which was confirmed by direct DNA sequencing (data not shown). The spaceflight also increased the level of cathepsin L mRNA about 2.4- and 2.0-fold in the P8 and P14 groups, respectively. The mRNA levels of µ-calpain, m-calpain, cathepsin B, and cathepsin H were not changed by the spaceflight.

In tail-suspended rats, the mRNA levels of components (RC2 and RC9) of 20S proteasome, E214K and polyubiquitin gradually increased from day 10 and reached levels 1.7-, 1.4-, 1.4-, and 2.8-fold higher than control values, respectively, on day 21 (Fig. 3B). The polyubiquitin transcripts in tail-suspended rats encoded repeated-ubiquitin moieties, as observed in spaceflight rats (data not shown). The expression of cathepsin L mRNA also significantly increased about 1.6-fold from day 5 to day 14 as compared with those of pair-fed control rats, but the level returned to the control value on day 21 (Fig. 3B). The expression of cathepsin B, cathepsin H, and m-calpain was unchanged during 3 wk of tail-suspension, but expression of µ-calpain mRNA decreased from day 10.

Effects of tail-suspension on muscle protease activities Sufficient amounts of gastrocnemius muscles were not available to analyze all of the protease activities in space samples. Therefore, we estimated the changes by precisely measuring protease activities in gastrocnemius muscle of tail-suspended rats. The peptidase activity of proteasome did not change until day 14 of suspension (Fig. 3B). On day 21, it significantly increased to 1.7-fold that in pair-fed control rats. Cathepsin B+L activity significantly increased in parallel with changes in the levels of expression of cathepsin L in suspended rats on day 5 and reached a maximum value on day 14. In contrast, calpain activity in gastrocnemius muscle was constant during suspension. Increase in ubiquitinated muscle proteins under microgravity conditions Because the level of polyubiquitin mRNA increased under microgravity conditions, we examined whether spaceflight and tail-suspension could induce the conjugation of ubiquitin to muscle proteins. The spaceflight augmented a 200-kDa ubiquitinated protein in gastrocnemius muscle of P8 rats (Fig. 4A), which was immunoreactive to a monoclonal antibody against rabbit skeletal fast-type MHC (data not shown). In addition to the MHC molecule, several ubiquitinated proteins with higher molecular masses (200–350 kDa) could be detected in the spaceflight rats, but not in the ground control rats. Proteins with molecular weights ranging from 120 to180 kDa were constantly ubiquitinated in both the ground control and spaceflight rats. In tail-suspended rats, ubiquitin-conjugated MHC first appeared on day 5 and continued to increase until day 14 (Fig. 4B). Tail-suspension also stimulated the conjugation of ubiquitin to muscle proteins (200–350 kDa) in a time-dependent manner. Effect of E-64 administration on MHC degradation Daily subcutaneous injection of E-64 at 4 and 8 mg/rat completely prevented the suspension-induced activation of cathepsin B+L in gastrocnemius muscle; however, it could not inhibit the fragmentation of MHC and failed to prevent the suspension-induced decreases in hindlimb muscle wet weights (Fig. 5), which suggests that the transient activation of cathepsin L may not play a critical role in the degradation of MHC observed in tail-suspended rats. DISCUSSION

The steady-state level of muscle myosin is maintained by regulating the synthesis of myosin, its assembly into thick filaments, and its subsequent degradation. Exposure to spaceflight has been shown to rapidly change MHC isoform expression from slow- (type I) to fast-type (type II) (20–22); however, the pathway by which fast-type MHC is degraded during spaceflight remains poorly understood. The present study showed that spaceflight and a model of spaceflight (tail-suspension) enhanced the ubiquitination and degradation of fast-type MHC in skeletal muscle of young developing rats. Myosin accounts for 45% of all myofibrillar protein in skeletal muscle, and muscle fiber exposed to spaceflight has been reported to occasionally exhibit loss of sarcomere structure with longitudinal streaming of Z-bands (23). Alternatively, it has been suggested that a small prolongation in MHC half-life could eventually lead to accumulation of this molecule in cultured cardiac muscle cells without any change in the rate of MHC synthesis (10). Thus, enhanced degradation of MHC may, at least in part, contribute to the muscle atrophy that arises during spaceflight. To identify specific proteases involved in degradation of MHC during spaceflight, we measured the level of expression of muscle proteases and related components at the mRNA level. At the two different ages (8- and 14-d-old rats at launch), the expression of proteasome components, polyubiquitin, and its conjugating enzyme was significantly up-regulated by the spaceflight. The most marked increase in expression was seen with polyubiquitin, and various proteins, including MHC, were ubiquitinated in atrophied gastrocnemius muscle in spaceflight rats. In tail-suspended rats, the level of ubiquitinated proteins also increased prior to the degradation of MHC or activation of proteasome. Eble et al. reported that lactacystin, a potent proteasome inhibitor, effectively suppressed the degradation of MHC in cultured cardiac myocytes (24). These findings suggest that, in rats sent into space, the ATP-ubiquitin-dependent proteolytic pathway may play an important role in degradation of muscle proteins. It is well known that starvation increases the mRNA levels of polyubiquitin and proteasome components (25). The shuttle rats in this mission lost more body weight than those in another space mission (STS-48) (11, 26), which indicates that their food intakes may have been insufficient. This finding might modify the mRNA levels of polyubiquitin and proteasome components in skeletal muscle. However, several investigations have reported that starvation decreased or did not affect the activities of cathepsins B, H, and B+L in skeletal muscle (27), whereas the expression of cathepsin L mRNA in gastrocnemius muscle of spaceflight rats was up-regulated by the spaceflight. Malnutrition also inhibits α-MHC isoform switching in cardiac muscle (28). However, the α-MHC isoform proteins were normally expressed in these shuttle rats, as described previously (12). We concluded that, based on these results, spaceflight—but not malnutrition—was primarily responsible for the stimulation of the ubiquitin-proteasome pathway. Solomon et al. have recently reported that MHC molecules did not function as effective substrates for the ubiquitin-proteasome proteolytic pathway, which suggests that structural alterations of MHC, such as cleavage by other proteases, may be required before ubiquitination and degradation by 26S proteasome (29). In addition to accumulation of the cathepsin L mRNA in spaceflight rats, we also found that tail-suspension significantly increased the cathepsin L mRNA level on day 10, when MHC degradation products started to accumulate, and muscle atrophy reached its peak. Therefore, we studied the effect of a potent inhibitor of cathepsin L,

E-64, to examine whether cathepsin L may have a role in the initiation of MHC degradation during unweighting. However, the inhibition of cathepsin L with E-64 failed to prevent the MHC degradation in vivo, which is consistent with other reports using in vitro systems (4, 30). This finding suggests that undefined modifications, other than limited processing by cathepsin L, may trigger the ubiquitination of MHC molecules under unweighting conditions. In fact, results obtained with models of microgravity conditions, such as immobilization and stretching, have suggested that oxidative stress or Ca2+-dependent phosphorylation may induce structural alterations of proteins, leading to ubiquitination (31, 32). Further studies are necessary to elucidate the mechanism that triggers ubiquitination of muscle proteins, including MHC, during space travel. Ubiquitin-protein ligase (E3) is a key enzyme in the ubiquitin-conjugation system (33). Recently, three independent investigations have suggested that c-Casitas B-lineage lymphoma (Cbl), an adapter protein, acted as a ubiquitin-protein ligase for several growth factor receptors, including epidermal growth factor receptor, and it down-regulates the signal pathway of growth factors (34–36). We performed differential display analysis with samples from this STS-90 mission and found that spaceflight significantly stimulated the expression of cbl-b, a Cbl family gene, in muscle (unpublished observation), which suggests that enhanced ubiquitination may also down-regulate the response of skeletal muscle cells to growth factors during spaceflight. There may be discrepancies between the space shuttle and tail-suspension experiments. We had to use newborn animals, because the present experiments were combined with other studies due to limitations in space shuttle experiments. However, newborn rats could not be used for tail-suspension experiments. We already reported that the ground newborn rats used in this study expressed the mRNA and protein of fast-type MHC molecules in fast-twitch skeletal muscles, similarly as 200-g body weight-rats did (12, 37). We also confirmed that the pattern of protease mRNA expression in gastrocnemius muscle was not significantly changed in normal rats from 8-d to 10-wk of age (data not shown). We suggest here that, base on these findings, both spaceflight and unweighting specifically enhance the degradation of fast-type MHC in association with activation of the ubiquitin-proteasome pathway. ACKNOWLEDGMENT We thank M. Yoshimoto, Taisho Pharmaceutical Co. (Tokyo, Japan) for a kind gift of E-64. This study was carried out as a part of “Ground Research Announcement for Space Utilization” promoted by Japan Space Forum. REFERENCES 1. Goldspink, D. F., Morton, A. J., Loughna, P. and Goldspink, G. (1986) The effect of

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Received October 4, 2000; revised January 3, 2001.

Table 1

Fig. 1

Figure 1. Hind-limb muscle wet weight of spaceflight (A) or tail-suspended rats (B). A) Gastrocnemius muscles of spaceflight rats were weighed and standardized by their body weights (mg/g body). B) Hind-limb muscles of suspended rats were isolated and weighed on the indicated days. Results were shown as the standardized muscle weight (mg/g body) in suspended rats relative to that in pair-fed control rats. Values are mean±SD (n = 6). *Significantly different, compared with the ground control rats or pair-fed, nonsuspended rats, P < 0.05.

Fig. 2

Figure 2. Accumulation of MHC degradation fragments in gastrocnemius muscle of spaceflight (A) or tail-suspended rats (B). Proteins (40 µg/lane) in a soluble fraction of gastrocnemius muscle were subjected to SDS-PAGE and transferred to a nylon membrane. Intact MHC (arrows) and its degradation fragments (closed triangles) were detected by Western blot analysis. MWSTD, molecular weights of standard.

Fig. 3

Figure 3. Protease mRNA expression in gastrocnemius muscle of spaceflight (A) and tail-suspension rats (B). Total RNA (1 µg) extracted from the indicated muscles was subjected to a semiquantitative RT-PCR analysis. mRNA concentrations were determined by calculating the ratio of the signal of the target cDNA to that of an internal standard β-actin cDNA (641 bp). Intensity of polyubiquitin cDNA encoding 2-repeated moieties was representative of polyubiquitin mRNA level. Activities of calpain, cathepsin B+L, and proteasome were also measured in gastrocnemius muscle of tail-suspended rats. Data are shown as the value in the suspended rats relative to that in the ground control rats or pair-fed, nonsuspended rats. Values are mean±SD (n = 6). *Significantly different, compared with the ground control rats or pair-fed, nonsuspended rats, P < 0.05.

Fig. 4

Figure 4. Accumulation of ubiquitinated proteins in gastrocnemius muscle of spaceflight (A) or tail-suspended rats (B). Proteins (40 µg/lane) in the soluble fraction of the muscle were subjected to SDS-PAGE by using a 6% polyacrylamide gel and transferred to a nylon membrane. Ubiquitinated proteins were detected by Western blot analysis. Arrows indicate ubiquitinated MHC. Similar results were obtained in three separate experiments. MWSTD, molecular weights of standard.

Fig. 5

Figure 5. Effects of E-64 on muscle cathepsin B+L activity (A), hind-limb muscle weight (B), and MHC fragmentation (C) in tail-suspended rats. A, B) E-64 (2 or 4 mg/rat) and vehicle (PBS) were subcutaneously injected into tail-suspended rats twice per a day (9:00 and 18:00) for 2 wk. The pair-fed and nonsuspended rats were also treated with vehicle. On day 14 of tail-suspension, hind-limb muscles were weighed, and cathepsin B+L activity in gastrocnemius muscle was measured. Values are mean±SD (n = 5). *, #Significantly different, compared with the pair-fed, nonsuspended rats or vehicle-treated, suspended rats, respectively, P < 0.05. C) Proteins (40 µg/lane) in soluble fraction of gastrocnemius muscle were subjected to SDS-PAGE and transferred to a nylon membrane. MHC (arrows) and its degradation fragments (closed triangles) were detected by Western blot analysis.