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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 26, 2005 as doi:10.1096/fj.04-3336fje. |
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,1
* Laboratoire de Physiologie des Systèmes Intégrés, CNRS UMR 6548, Nice, France; and
Laboratoire de Physiologie des Adaptations, Performance Motrice et Santé, Université de Nice, Nice, France
1 Correspondence: Laboratoire de Physiologie des Adaptations, Performance Motrice et Santé, Université de Nice - UFR STAPS, 261 route de Grenoble, Nice 06205, France. E-mail: achopard{at}unice.fr
SPECIFIC AIMS
Skeletal muscle fibers must withstand mechanical constraints exerted on their longitudinal and transversal axes because of force generation and external loading (Fig. 1
A). The variable mechanical constraints cause structural differences on the longitudinal axis of slow and fast muscle fibers, in the myotendinous junction (MTJ), and in Z lines. On the transversal axis, we paid special attention to dystrophin and its associated proteins (DGC) and to several proteins of specific sites at the level of the Z disk, that is "costamere" (vinculin-talin-intregrin system). These two systems mediate force transduction from myofibrils to extracellular matrix across the sarcolemma-associated cytoskeleton, stabilizing the cell membrane during activity (Fig. 1C
). Many studies have focused on these proteins that are involved in several myopathies and that play an important role in muscle fiber structure, cell integrity maintenance, signaling, or force transmission.
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This study was designed to evaluate the effects of hypokinesia and hypodynamia on cytoskeletal and related protein contents from human skeletal muscle. Twelve proteins (dystrophin and its associated proteins, dysferlin, talin, vinculin, and metavinculin, 
-actinin, desmin, actin, and myosin), including DGC and costameres, were quantitatively analyzed during an 84 day long-term bedrest (LTBR). The preventive-compensatory effects of maximal resistance exercise (MRE) were evaluated as a countermeasure.
PRINCIPAL FINDINGS
1. Comparison of cytoskeletal and related protein contents between human SOL and VL
Cytoskeletal and related protein contents were higher in VL than in SOL (12–94%, P<0.05). In previous studies on rat muscle, we reported cytoskeletal protein contents were higher for SOL than for EDL. We interpreted these differences as corresponding to a physiological response of the muscle fibers to duration, magnitude, and frequency of the imposed mechanical loading. This interpretation is reinforced by the present study on human muscle. Their architecture largely explains the differences between SOL and VL in maximal force and velocity of shortening of the individual fibers and of the whole muscle: SOL fibers exhibit less tension with respect to the main axis of their tendon of insertion and less velocity of shortening potential than VL fibers. Moreover, force transmission appears not to be limited to the myotendinous junction, and lateral force transmission is to be considered, which may differ for muscles of different degrees of pennation.
The importance of DGC in muscle fiber cohesion and in protection of the sarcolemma from contraction-induced damage was demonstrated in various congenital muscular dystrophies in human or animal models. The differences observed between SOL and VL could be explained by the differential mechanical constraints imposed on their fibers (i.e., cytoskeletal protein contents increase with mechanical constraints). The VL fibers must frequently resist higher mechanical constraints than do SOL fibers.
Vinculin is a major component of the costameres, and it was found within the Z-disk in striated muscle, participating in the molecular bridges joining the Z lines to extracellular matrix. Metavinculin appears to be involved in intercellular junctions: adherent junctions in smooth muscle and intercaled disks in cardiac muscle. A low level of metavinculin was found in sartorius muscle, which our results confirm in human muscle. Results showed a higher content of both proteins in the VL, particularly for metavinculin (+94%, P<0.01). These differences are comparable to those we reported for rat muscles where metavinculin was not present in control SOL but largely present in EDL.
Mechanisms other than those involved in the resistance of the cytoskeleton to mechanical constraints may be implicated. For example, 
-sarcoglycan is a member not only of the transplasmalemmal DGC but also of the SR over the I-bands, including terminal cisternea, and it was suggested that: 1) this protein may be related to calcium metabolism; and 2) it may anchor the SR to the myofilaments during muscle contraction and relaxation. These two notions may account for the higher differences (+44%, P<0.01) in VL than in SOL for -sarcoglycan than for the other members of DGC.
This first quantification of dysferlin in human muscle showed a higher content in VL than in SOL (+29%, P<0.05). This membrane-associated protein, which is not linked to DGC has been suggested to be involved in membrane fusion or repair. The increased intracellular Ca2+ concentration sets in motion a sequence of events among which dysferlin is thought to act as a Ca2+-dependent "hook" that will enable the efficient fusion of the repair patch with the sarcolemma. However, its function is not completely known.
2. Effect of LTBR on skeletal muscle fiber cross sectional area (CSA), myosin expression, and cytoskeletal and related proteins
Structural alterations in muscle submitted to unloading (simulated or actual microgravity) are illustrated first by muscle atrophy. After LTBR we observed a decrease of 28 and 29%, respectively, for slow twitch (ST) and fast twitch (FT) CSA in SOL, and of 17% for ST CSA in VL (P<0.1). A shift of fiber phenotype from ST to FT is generally observed in disused atrophied muscle. Although expression of slow MHC tended slightly to decrease, we found no significant alterations in MHC expression for VL after LTBR. The 40% increase in fibers expressing fast MHC, which is indicative of the effects of LTBR on postural muscles, was not significant because of the large variability in MHC expression among subjects and the small number of biopsies obtained for SOL.
Actin and myosin decreased in VL, respectively, by 9 and 14% (P<0.05), and the same tendency was observed in SOL. Data on animals indicate such a decrease in contractile proteins, suggested also by a few results obtained on humans, during spaceflight of short duration. This decrease appears to result from muscle disuse.
After LTBR, proteins belonging to the DGC, dysferlin, and proteins of the costamere exhibited large increases, higher in SOL (67–216%) than in VL (32–142%) (Fig. 2
). It is suggested that the DGC functions in order to anchor the sarcolemma to costameres. Plasma membrane remodeling during muscle atrophy is probably one of the key points for interpreting the increases in DGC relative content in SOL and in VL that result from LTBR. Membrane remodeling probably also accounts for the increase in talin relative content in VL (+60%, P<0.01). This protein for which specific sites in force transmission are enriched at MTJ participates in the link between peripheral Z-disk and extracellular matrix, within the costameres, with other focal adhesion proteins like vinculin, -actinin, and ß1-integrin. The large increase in costameric metavinculin level (+213%, P<0.01) in human SOL is in line with the de novo expression of this protein we reported in rat SOL after hindlimb suspension.
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As mentioned previously, mechanisms other than those involved in the resistance of the cytoskeleton to mechanical constraints may be implicated in the reported changes. This is illustrated by -sarcoglycan, which exhibited the highest increase (216% in SOL and 142% in VL, P<0.01). Studies revealed a proliferative effect of unloading on the SR in SOL, in line with a more developed SR in fast twitch fibers. This increase in SR and its timing could be related to the large increase in -sarcoglycan content after LTBR, which will be partly a companion change of cellular structures involved in calcium regulation.
Although DGC and proteins of the costameres largely contribute to maintaining plasma membrane integrity, physical injuries of plasma membrane could occur during muscle cell contraction or stretch. Defects in plasma membrane repair could cause muscle degeneration. As dysferlin appears to be involved in mechanisms for membrane plasma repair, the increase in dysferlin after unloading (from 104%, P<0.1 in SOL, and 71%, P<0.01 in VL) could be interpreted as being linked with this function. It could be postulated that the increased content of dysferlin would preserve the capacity of a muscle either impaired or submitted to increased mechanical constraints to be able to face any damages resulting from functional demands.
3. Effects of maximal resistance exercise as countermeasure
During this LTBR, MRE partly prevented muscle atrophy and mostly the protein content changes. After LTBR with MRE, muscle fiber CSA did not decrease in SOL, but in VL the decrease in ST fiber CSA was still present (14%, P<0.01). Concerning MHC expression in SOL, we observed a significant 13% decrease and a 62% increase (P<0.01), respectively, for fibers expressing slow MHC and fast MHC. These findings suggest that MRE, which seeks FT fibers preferentially, accentuated the expression of fast myosin in SOL.
Most changes in cytoskeletal protein contents that resulted from LTBR were compensated by MRE in postural SOL (Fig. 2)
. Only -sarcoglycan and dysferlin contents were still increased by 70% and 108%, respectively (P<0.1). This could be considered the companion changes of the changes induced in muscle profile (fast MHC expression; enriched SR in the FT fibers).
MRE was not able to compensate totally the changes in cytoskeletal protein contents induced by LTBR in VL (Fig. 2)
as it did not compensate ST fiber atrophy. The decreases in myosin and actin contents after LTBR were compensated by MRE. On the other hand, even if the increase was lower in VL than after LTBR without MRE, dystrophin, ß-dystroglycan, -sarcoglycan, -sarcoglycan, dysferlin, and talin contents were still higher (28–132%, P<0.05). Vinculin and metavinculin, which exhibited no significant change in VL after LTBR, were increased with MRE during LTBR, reinforcing the pre-LTBR differences between SOL and VL. As for SOL, dysferlin remained largely increased in VL (+132%, P<0.01), a mechanism that might strengthen cellular membrane repair, because of exercise intensities. No other data are available for comparison.
Finally, the changes observed in VL were lower with MRE than after LTBR only, which suggests that MRE, which imposes a more usual type of tension for this mixed muscle, was not sufficient to prevent the induced changes for DGC proteins, although it is able to exacerbate the differences observed between VL and SOL for dysferlin and costamere proteins.
CONCLUSIONS AND SIGNIFICANCE
The differences in cytoskeletal protein relative contents between fast and slow muscles, the changes resulting from a reduction of the load imposed on muscles, and the fairly positive effects of maximal resistance exercise as a countermeasure illustrate the importance of these proteins in a structure-function relation in muscles, either in force transmission, cell integrity, or cellular repair (Fig. 1)
. This knowledge will contribute to the development of efficient space flight countermeasures and rehabilitation methods in clinical situations where musculoskeletal unloading is a component. The results on individual cytoskeletal and related proteins should serve in understanding these protein functions and their roles in muscle fiber physiology and physiopathology.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3336fje; doi: 10.1096/fj.04-3336fje
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P < 0.01 vs. control (pre-bedrest values = 100 in arbitrary units).