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.

We have previously reported that spaceflight and tail suspension enhanced degradation of rat myosin heavy chain (MHC) in association with activation of a ubiquitin-dependent proteolytic pathway [Ikemoto et al., FASEB J. 15 (2001), 1279-1281]. To elucidate whether the ubiquitination is accompanied by oxidative stress, we measured markers for oxidative stress, such as thiobarbituric acid-reactive substance (TBARS) and glutathione disulfide (GSSG), in gastrocnemius muscle of tail-suspended rats. Glutathione (GSH) concentration in the muscle significantly decreased from day 5 and reached a minimum value on day 10. Tail suspension reciprocally increased concentrations of TBARS and GSSG in parallel with enhancement of protein ubiquitination, suggesting that oxidative stress may play an important role in protein ubiquitination caused by tail suspension. To prevent ubiquitination associated with oxidative stress, we also administered an antioxidative nutrient, cysteine, to tail-suspended rats. Intragastric supplementation of 140 mg/rat of cysteine for 2 weeks or longer normalized the ratio of GSH to GSSG in the muscle and suppressed protein ubiquitination and MHC fragmentation, compared with supplementation of the equimolar amount of alanine. The cysteine supplementation significantly suppressed the loss of hindlimb muscle mass. Our results suggest that supplementation of antioxidative nutrients, such as cysteine, may be beneficial for preventing ubiquitination of muscle proteins caused by unweighting.

No abstract

Most teleostean embryos develop and hatch without parental assistance, though some receive parental care. We focused on a paternal brood‐care species, the barred‐chin blenny (Rhabdoblennius nitidus [Günther, 1861]). As hatching approached, fanning behavior by the male parent drastically increased and then embryos hatch. In the absence of the male parent, most embryos failed to hatch. However, the hatching rate was greatly assisted by introducing an artificial water current, suggesting that paternal assistance other than for aeration is required for successful embryo hatching. Next, we analyzed genes for the hatching enzyme and egg‐envelope protein, which were successfully cloned from barred‐chin blenny, and found the expression patterns differed from those of other euteleosts. Generally, high choriolytic enzyme swells the intact egg envelope, and then low choriolytic enzyme solubilizes the swollen envelope. The expression levels of both the enzymes, but especially the latter, were much lower in barred‐chin blenny that is known in most other oviparous species. In addition, the main component of the egg envelope was changed into ChgHm and choriogenin L (ChgL) in barred‐chin blenny, whereas ChgH and ChgL for other euteleosts. These in barred‐chin blenny would result in ineffective egg‐envelope digestion because the posthatching egg envelopes were observed to be swollen but not solubilized. Male parental assistance by fanning until hatching may compensate for this insufficiency. Our study illustrates an example of the evolution of parent‐embryo interaction built on a novel relationship: Degradation of the hatching enzyme/egg‐envelope digestion system, accompanied by male parental hatching assistance.

In this study, we examined the effects of a potent cysteine protease inhibitor, N-(L-3-transcarboxyoxirane-2-cabonyl)-L-leucine-4-aminobutylamide (E-64a), on bone weight and strength in tail-suspended rats. We first administered a vehicle or 4 or 8 mg/rat of E64a to rats fed with a low calcium diet for 7 wks to determine effective doses of E-64a on bone resorption in vivo. Femoral cathepsin K-like activity and serum hydroxyproline level in rats fed with a low calcium diet were significantly higher than those in rats fed with a standard diet. The intraperitoneal injection of 8 mg/rat of E-64a to rats decreased their serum calcium and hydroxyproline concentrations after 3 to 6 hrs in parallel with changes in femoral cathepsin K-like activity, while 4 mg/rat of E-64a had weaker effects on these parameters. Based on these results, we injected 8 mg/rat of E-64a to tail-suspended rats twice a day for 2 wks and compared the results with those of treatment with 1 mg/rat of etidronate, a bisphosphonate, twice a week. In tailsuspended rats, femoral weight and strength, assessed by three-point bending test, significantly decreased from Day 5 to 21, while femoral cathepsin K-like activity and serum calcium and hydroxyproline concentrations did not change. E-64a inhibited femoral cathepsin K-like activity in tail-suspended rats, but etidronate did not. E64a as well as etidronate significantly prevented the suspension-induced declines in bone weight and strength. However, more frequent injection and higher doses were required for E-64a to exhibit significant efficacy of antiresorption, compared with those of etidronate. Our results suggest that a cysteine protease inhibitor could improve suspension-induced osteopenia by inhibiting cathepsin K-like activity in bone; however, it needs several improvements in the effect as a clinical drug.