| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 12, 2001 as doi:10.1096/fj.00-0629fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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; and
¶ Division of Enzyme Chemistry, Institute for Enzyme Research, the University of Tokushima, Tokushima 770-8503, Japan
2Correspondence: Department of Nutrition, School of Medicine, The University of Tokushima, 3–18-15 Kuramoto-cho, Tokushima 770-8503, Japan. E-mail: nikawa{at}nutr.med.tokushima-u.ac.jp
SPECIFIC AIMS
To elucidate the mechanism of muscle atrophy induced by space travel, we examined the degradation of a fast-type contractile myosin heavy chain (MHC) protein and expression of key enzymes or components in the three major proteolytic pathways (Ca2+-dependent, lysosomal, and ubiquitin–proteasome pathways) in skeletal muscle of young rats subjected to spaceflight (STS-90). We report here that atrophied gastrocnemius muscle is specifically sensitive to the ubiquitin–proteasome proteolytic pathway.
PRINCIPAL FINDINGS
1. Spaceflight led to significant accumulation of MHC degradation
products in gastrocnemius muscle
In the STS-90 mission, the flight rats were launched into space in
a space shuttle, when they were 8 (P8) and 14 days old (P14),
respectively, and returned to the Kennedy Space Center after a 16 day
stay in space.
The levels of a fast-type 200 kDa MHC protein in gastrocnemius muscle
of both the P8 and P14 spaceflight rats were lower than those of the
respective ground control rats (Fig. 1A
). 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, since incubation of
purified MHC with papain produced fragments of the same molecular
sizes.
|
The spaceflight-induced degradation of MHC could be replicated in a
model of microgravity conditions (tail suspension). Tail suspension for
10 days or longer caused the accumulation of immunoreactive degradation
products of MHC, and the most severe degradation of MHC was observed on
the last day of tail suspension (day 21), as shown in Fig. 1B
.
2. Spaceflight increased mRNA levels of cathepsin L, proteasome
components, polyubiquitin, and ubiquitin-conjugating enzyme
(E214K) in gastrocnemius muscle
To identify the proteases that mediate the degradation of MHC
during spaceflight, the expression of muscle protease and its related
components was analyzed by semiquantitative reverse
transcription-polymerase chain reaction (RT-PCR). The expression of
components (RC2 and RC9) of 20S proteasome, E214K
and polyubiquitin was up-regulated at the mRNA level by the
spaceflight, compared with the respective ground control rats
(Fig. 2A
). The spaceflight increased the levels of polyubiquitin
mRNA encoding 2-, 3-, 4-, 5-, and 7-repeated ubiquitin moieties in
tandem (indicated by open arrowheads in Fig. 2A
), which was
confirmed by direct DNA sequencing. The spaceflight also increased the
levels 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 expression 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. 2B
). The polyubiquitin transcripts in tail-suspended rats
encoded repeated ubiquitin moieties, as observed in spaceflight rats.
The expression of cathepsin L mRNA also significantly increased about
1.6-fold from day 5 to 14 as compared with those of pair-fed control
rats, but the level returned to baseline on day 21. In contrast, the
level of expression of cathepsin B, cathepsin H, and m-calpain was
unchanged during 3 wk of tail suspension, and the expression of
µ-calpain mRNA was suppressed on day 10 and later. The activities of
proteasome and cathepsin B+L changed in tail-suspended rats in parallel
with increases in the mRNA levels of proteasome components and
cathepsin L, respectively.
3. Spaceflight led to accumulation of ubiquitinated muscle
proteins, including a MHC molecule in gastrocnemius muscle
Since 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, which was immunoreactive to a monoclonal antibody against rabbit
skeletal fast-type MHC. 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. In tail-suspended rats, ubiquitin-conjugated MHC first
appeared on day 5 and continued to increase until day 14. Tail
suspension also stimulated the ubiquitin conjugation of the muscle
proteins (200–350 kDa) in a time-dependent manner.
4. Administration of a cysteine protease inhibitor, E-64,
to the suspended rats did not prevent the MHC degradation
In addition to accumulation of the cathepsin L mRNA in
spaceflight rats, we also showed 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. To
examine whether cathepsin L may have a role for the initiation of MHC
degradation during unweighting, we injected a potent inhibitor of
cathepsin L, E-64, to tail-suspended rats. 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.
CONCLUSIONS
The loss of muscle mass and strength, particularly in antigravity slow-twitch muscles, is a major complication for the crews of spacecraft; however, the mechanisms that cause the atrophy are not fully understood.
The present study showed that spaceflight and tail suspension enhanced the degradation and ubiquitination of fast-type MHC in skeletal muscle of young developing rats. Myosin accounts for 45% of all myofibrillar proteins 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. 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. Thus, enhanced degradation of MHC may contribute at least in part to the muscle atrophy that arises during spaceflight.
There is still controversy with regard to the potential roles of the
major proteolytic pathways in muscle wasting. We have precisely
determined the expression of key enzymes or components in the
proteolytic pathways in the spaceflight rats and found that spaceflight
stimulated the ubiquitination of muscle proteins, including MHC, and
stimulated the expression of ubiquitin–proteasome pathway genes, but
had no effect on the lysosomal and calpain-dependent pathways. Using a
microgravity model (tail-suspended rats), we also confirmed that
unweighting specifically activated the ubiquitin–proteasome-dependent
pathway. Our results provide evidence for the first time that the
ubiquitin–proteasome-dependent pathway in muscle is sensitive to
spaceflight (Fig. 3
).
|
MHC molecules did not function as effective substrates for the ubiquitin–proteasome pathway, suggesting that structural alterations, such as cleavage by other proteases, may be required before ubiquitination and degradation by 26S proteasome. We therefore focused on the transient activation of cathepsin L in tail-suspended rats. However, the inhibition of cathepsin L with E-64 failed to prevent the MHC degradation in tail-suspended rats, suggesting 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 have suggested that oxidative stress or Ca2+-dependent phosphorylation may induce structural alterations of proteins, leading to ubiquitination. Elucidation of the pathway that triggers ubiquitin-conjugation during spaceflight is the next important step toward understanding muscle atrophy associated with space travel.
Recently, c-Casitas B-lineage lymphoma (Cbl), an adapter protein, has
been reported to act as a ubiquitin-protein ligase (E3) for several
growth factor receptors, including epidermal growth factor receptor,
and to down-regulate the signaling pathway of growth factors. We
performed differential display analysis using 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), suggesting that enhanced ubiquitination may also
down-regulate the response of skeletal muscle cells to growth factors
during spaceflight (Fig. 3)
. Thus, ubiquitination of muscle proteins
may be an essential step to accommodate skeletal muscle to weightless
conditions.
In conclusion, the present results provide not only a new insight into the mechanism for muscle atrophy in disuse as well as during space travel, but also important information for the prevention and management of the atrophy.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0629fje ; to cite this article, use FASEB J. (March 12, 2001) 10.1096/fj.00-0629fje 
This article has been cited by other articles:
|
|
 |
|
  K. Hirasaka, S. Kohno, J. Goto, H. Furochi, K. Mawatari, N. Harada, T. Hosaka, Y. Nakaya, K. Ishidoh, T. Obata, et al. Deficiency of Cbl-b Gene Enhances Infiltration and Activation of Macrophages in Adipose Tissue and Causes Peripheral Insulin Resistance in Mice Diabetes, October 1, 2007; 56(10): 2511 - 2522. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Kawano, Y. Matsuoka, Y. Oke, Y. Higo, M. Terada, X. D. Wang, N. Nakai, H. Fukuda, S. Imajoh-Ohmi, and Y. Ohira Role(s) of nucleoli and phosphorylation of ribosomal protein S6 and/or HSP27 in the regulation of muscle mass Am J Physiol Cell Physiol, July 1, 2007; 293(1): C35 - C44. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Vermaelen, P. Sirvent, F. Raynaud, C. Astier, J. Mercier, A. Lacampagne, and O. Cazorla Differential localization of autolyzed calpains 1 and 2 in slow and fast skeletal muscles in the early phase of atrophy Am J Physiol Cell Physiol, May 1, 2007; 292(5): C1723 - C1731. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Landsverk, S. Li, A. H. Hutagalung, A. Najafov, T. Hoppe, J. M. Barral, and H. F. Epstein The UNC-45 chaperone mediates sarcomere assembly through myosin degradation in Caenorhabditis elegans J. Cell Biol., April 23, 2007; 177(2): 205 - 210. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Kalb and D. Solomon Space Exploration, Mars, and the Nervous System Arch Neurol, April 1, 2007; 64(4): 485 - 490. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Urso, Y.-W. Chen, A. G. Scrimgeour, P. C. Lee, K. F. Lee, and P. M. Clarkson Alterations in mRNA expression and protein products following spinal cord injury in humans J. Physiol., March 15, 2007; 579(3): 877 - 892. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Oguro, T. Sakurai, Y. Fujita, S. Lee, H. Kubota, K. Nagata, and Y. Atomi The molecular chaperone HSP47 rapidly senses gravitational changes in myoblasts Genes Cells, November 1, 2006; 11(11): 1253 - 1265. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Urso, A. G. Scrimgeour, Y.-W. Chen, P. D. Thompson, and P. M. Clarkson Analysis of human skeletal muscle after 48 h immobilization reveals alterations in mRNA and protein for extracellular matrix components J Appl Physiol, October 1, 2006; 101(4): 1136 - 1148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. E. Dupont-Versteegden, J. D. Fluckey, M. Knox, D. Gaddy, and C. A. Peterson Effect of flywheel-based resistance exercise on processes contributing to muscle atrophy during unloading in adult rats J Appl Physiol, July 1, 2006; 101(1): 202 - 212. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Cieniewski-Bernard, Y. Mounier, J.-C. Michalski, and B. Bastide O-GlcNAc level variations are associated with the development of skeletal muscle atrophy J Appl Physiol, May 1, 2006; 100(5): 1499 - 1505. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Morris, L. D. Morris, A. R. Kennedy, and H. L. Sweeney Attenuation of skeletal muscle atrophy via protease inhibition J Appl Physiol, November 1, 2005; 99(5): 1719 - 1727. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ogawa, T. Nikawa, H. Furochi, M. Kosyoji, K. Hirasaka, N. Suzue, K. Sairyo, S. Nakano, T. Yamaoka, M. Itakura, et al. Osteoactivin upregulates expression of MMP-3 and MMP-9 in fibroblasts infiltrated into denervated skeletal muscle in mice Am J Physiol Cell Physiol, September 1, 2005; 289(3): C697 - C707. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Yimlamai, S. L. Dodd, S. E. Borst, and S. Park Clenbuterol induces muscle-specific attenuation of atrophy through effects on the ubiquitin-proteasome pathway J Appl Physiol, July 1, 2005; 99(1): 71 - 80. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Fluck, S. Schmutz, M. Wittwer, H. Hoppeler, and D. Desplanches Transcriptional reprogramming during reloading of atrophied rat soleus muscle Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2005; 289(1): R4 - R14. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. B. Reid Response of the ubiquitin-proteasome pathway to changes in muscle activity Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2005; 288(6): R1423 - R1431. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. C. DeRuisseau, A. N. Kavazis, M. A. Deering, D. J. Falk, D. Van Gammeren, T. Yimlamai, G. A. Ordway, and S. K. Powers Mechanical ventilation induces alterations of the ubiquitin-proteasome pathway in the diaphragm J Appl Physiol, April 1, 2005; 98(4): 1314 - 1321. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. J. Stevenson, A. Koncarevic, P. G. Giresi, R. W. Jackman, and S. C. Kandarian Transcriptional profile of a myotube starvation model of atrophy J Appl Physiol, April 1, 2005; 98(4): 1396 - 1406. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Powers, A. N. Kavazis, and K. C. DeRuisseau Mechanisms of disuse muscle atrophy: role of oxidative stress Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2005; 288(2): R337 - R344. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. W. Jackman and S. C. Kandarian The molecular basis of skeletal muscle atrophy Am J Physiol Cell Physiol, October 1, 2004; 287(4): C834 - C843. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. C. Harrison, D. L. Allen, B. Girten, L. S. Stodieck, P. J. Kostenuik, T. A. Bateman, S. Morony, D. Lacey, and L. A. Leinwand Skeletal muscle adaptations to microgravity exposure in the mouse J Appl Physiol, December 1, 2003; 95(6): 2462 - 2470. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. DeFelipe, J.I. Arellano, A. Merchan-Perez, M.C. Gonzalez-Albo, K. Walton, and R. Llinas Spaceflight Induces Changes in the Synaptic Circuitry of the Postnatal Developing Neocortex Cereb Cortex, August 1, 2002; 12(8): 883 - 891. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
