This study describes the morphologic changes in rabbit soleus muscle following hindlimb suspension (HS) for 1 to 4 weeks (group A); or following HS with hindfeet passively dorsiflexed, by means of an elastic band, for 1 to 2 weeks (group 6). In the latter, elastic band use allowed phasic contractions of foot extensor muscles against resistance and prevented 35% chronic soleus shortening, which occurred in group A animals. In group A, the soleus revealed progressive muscle atrophy and myofibrillar damage. Myofibrils underwent dissolution, muscle regeneration was ineffective, and adipose tissue developed from about 2-week suspension onward. Conversely, passive dorsiflexion of unloaded hindfeet was essential in maintaining mass and structural muscle integrity in the soleus of group B. It is hereby demonstrated that HS-induced soleus damage in the rabbit is progressive, and can be prevented, avoiding long-term shortening of soleus and its phasic unloaded contractions. Soleus sensitivity to unloading conditions, such as HS, tenotomy, and hypogravity, may depend on the particular physiology of this tonic antigravity muscle, engaged mainly in developing long-lasting isometric contractions in a stretched length. 0 1992 John Wiley & Sons, Inc. Key words: hindlimb suspension soleus muscle myofibrillar disruption muscle length muscle atrophy MUSCLE & NERVE 15:1002-1015 1992
ACTIVE MUSCLE LENGTH REDUCTION PROGRESSIVELY DAMAGES SOLEUS I N HINDLIMB-SUSPENDED RABBITS GIUSEPPE SANCESARIO, MD, ROBERTO MASSA, MD, ARCHINTO P. ANZIL, MD, and GlORGlO BERNARDI, MD
Hindlimb suspension techniques have been increasingly used as a simple and inexpensive ground-based model in order to reproduce some effects of weightlessness on antigravity muscles of small mammals. HS in experimental animals as well as microgravity in astronauts eliminate hindlimb-supported body weight. It seems obvious that antigravity muscles produce little or no force under these conditions. ' 2 , L 7 Unexpectedly, chronically recorded electromyograms in rats show almost normal levels of electrical activity in the unloaded muscles, particularly in the tibialis anterior, the soleus, and the gastrocnemius, during From the Laboratory of Experimental Neuropathology. Institute of Neurology, Second University of Rome, Italy (Drs Sancesario, Massa. and Bernardi); and the Department of Pathology, SUNY-HSCE, Brooklyn, New York (Dr. Anzil). Acknowledgments. This study was supported in part by a grant from the Agenzia Spaziale ltaliana (AS1 90-RS-30). Address reprint requests to Dr Giuseppe Sancesario, Laboratory 01 Experimental Neuropathology, Institute of Neurology, Second University of Rome, Via 0.Raimondo 8, 00173 Rome, Italy. Accepted lor publication February 3, 1992 CCC 0148-639X1921091002-14 $04.00 0 1992 John Wiley & Sons, Inc.
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'
prolonged HS. Moreover, the contractile activity of the soleus, a tonic muscle, shifts to a phasic pattern after the fourth day of HS." In spite of persistent muscle activity, up to 50% reduction in muscle weight arid pertinent morphologic changes have been reported to develop in the soleus of the rat, mouse, and hamster after the first week of ~ ~ . 5 , 1 7 , 2Although 1 conditions of hypokinesiahypodynamia obviously affect suspended hindlimbs,"*26 the mechanism by which unloading of the hindlimbs specifically damages the soleus fibers is not clear. In recent experiments, we have observed that HS of rabbits for 1 week produces, in soleus myofibers, multifocal areas of myofibrillar disruption concomitant with muscle atrophy.* T h e myofibrillar- lesions were thought to be dependent on both the conditions of chronic shortening and periodic contraction against no load occurring in suspended soleus.27'L2Similar conclusions have recently been drawn from experiments in HS of rats. 'Lo With this in mind we can hypothesize that segmental loss of sarcomeres in the soleus during the first week of HS may represent a cytoarchitectural
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remodeling of the myofibers in an attempt to adjust to their new and shorter working length.3 Alternatively, we can presume that phasic and tonic soleus contractions without a load, if they are long-lasting, may constitute a persistent detrimental factor, leading to widespread muscle damage. Either way, the abnormal shortening of the soleus would play a major part in causing myofibrillar changes. The present study was designed to investigate whether the segmental myofibrillar damage induced by HS in the soleus is a self-limiting or a worsening process, roughly proportional to the duration of suspension. In addition, we attempted to prevent the occurrence of myofibrillar disruption in the soleus by avoiding the reduction of muscle length during HS. Our approach was to apply an elastic resistance to the plantar flexion of the feet of suspended rabbits. In this way, the unloaded hindlimbs could be actively extended against resistance, while postural changes determined by suspension were prevented. Data are presented on the severe damage to myofibers determined in the soleus muscle by prolonged HS. Furthermore, we report here on the finding that the resistance imposed on chronic shortening and on phasic soleus muscle contractions is essential in maintaining mass and structural fiber integrity during unloading of the hindlimbs. Appropriate muscle length and resistance to contraction may represent the specific biomechanical requirements for the physiology of the antigravity soleus muscle, engaged mainly in developing long-lasting isometric contractions. ANIMALS AND METHODS
The experiments were performed on young, male New Zealand rabbits (1025 to 1150 g body weight), randomly assigned to three groups: ground-based control (group C, n = 24), hindlimb suspended (group A, n = 24), and hindlimb suspended plus passive dorsiflexion of the feet (group B, n = 16). Animal experimentation was conducted in accordance with the institution's guide for the care and use of laboratory animals. Control rabbits were kept in standard laboratory conditions. Experimental rabbits were suspended in a horizontal position as previously reported.2z22In brief, the animals were dressed with a denim harness and were suspended from a hook so that, while forelimbs stood on a wooden support, hindlimbs dangled down freely for 1, 2, or 4 weeks. Animals in group B were also suspended
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according to this model for 1 or 2 weeks; however, a dorsiflexed posture of the feet at 45" was passively maintained with a rubber band fastened to the foot at one end and to the denim harness at the other end. The length of the rubber band and its fastening to the harness were selected so that unloaded hindlimbs were kept flexed as in the control animals at rest on the floor of the cage. The resistance of the rubber band was empirically chosen so that animals could extend their hindlimbs without moving their body wrapped in the harness. In stretching the rubber band by 6 cm, the ankle joint angle could be opened from 45" to go", which is the estimated range of ankle movement in walking rabbits before HS. A weight of 270 to 280 g, comparable to 21% to 26% of the body weight of suspended animals, was evaluated to be sufficient to stretch an extremity of the rubber band by 6 cm. Body weight was assessed before and after the period of suspension. Food intake was measured daily. The behavior of the suspended animals was also observed daily for 30 minutes in the morning and 30 minutes in the afternoon. At the end of a given suspension period, the animals were immediately sacrificed under barbiturate/ketamine anesthesia; care was taken to avoid reloading the hindlimbs in removing the animals from the suspension apparatus. A t each stage of the experiment 3 animals from each group were used. The solei were excised, the midbelly regions were mounted in tragacanth gum and quickly frozen in isopentane cooled in liquid nitrogen. Cryostat sections (10 Fm) were stained either for modified Gomori's trichrome, alkaline and acid myofibrillar adenosinetriphosphatase (ATPase), nicotinamide adenine dinucleotide dehydrogenase (NADH-d), or for Oil Red 0. The type of fibers, and the numbers of necrotic or regenerating ones, were evaluated in control and suspended animals in at least 200 fibers from randomly selected fields of each muscle in pertinent histological and histochemical sections. Histochemistry.
Five animals from each group were used at each stage of the experiment. The hindquarters were perfused through the abdominal aorta with a 4% glutaraldehyde solution in 0.1 mol/L phosphate buffer, pH 7.4. After perfusion, the soleus muscles were removed, weighed, and kept in cold perfusate overnight. Under a stereomicroscope several samples, ap-
Electron Microscopy.
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also occasionally extended their hindlimbs u p to 100" against the resistance created by the rubber band. Food intake was about 20% less in groups A and B animals for a few days; afterward, it was the same as for group C animals. However, the gain in body weight was significantly reduced after the first week of suspension in groups A and B (Table 1). The wet weight of the soleus in group A animals was 18.6%, 47.6%, and 41.9% less than in control animals after 1, 2, and 4 weeks, respectively. On the contrary, in group €3 animals, the wet weight of the soleus was not significantly different from that of control animals after 1 and 2 weeks, respectively (Table 1). Compared with control animals, the soleus weighdbody weight ratio was significantly lower in group A, whereas, in group €3, it was equal at 1-week and even higher after 2-week suspension.
proximately 2 x 2 mm, were cut from the midbelly region of the muscle. T h e pieces were rinsed in buffer, postfixed for 90 minutes in 1% buffered osmium tetroxide (at 20" C) and conventionally embedded in Epon 812. Semithin sections were cut according to a transverse or longitudinal plane with an LKB ultramicrotome and stained with paraphenylenediamine. They were viewed and photographed with a phase contrast Leitz automatic photomicroscope. Thin (silver) sections were stained with uranyl acetate and lead citrate and examined with a Zeiss EM-10 electron microscope. T h e number of fibers with a recognizable sarcomeric banding pattern was evaluated in random fields after scoring an average of 250 fibers per muscle in longitudinal semithin sections. T h e morphometric evaluation was made using a MOP-3 image analysis system (Zeiss) on photographic prints of muscle semithin transverse sections. 'The mean cross fiber area was calculated in random fields of soleus from both sides of each animal, after measuring an average of 250 30 fibers per muscle. Because of the clear-cut difference between the two experimental groups, the comparison was limited to the results obtained at 1- and 2-week suspension. Soleus weight to body weight ratios and measurements of cross fiber areas were analyzed using the one-way ANOVA test.
*
Muscle fibers with moderately high NADH-d activity predominated in the control rabbit soleus. Fibers staining dark with acid ATPase reaction represented 80% to 85% of' the fiber population. Group A Animal.\. 'Ihe longer the period of suspension, the greater the changes observed in soleus fibers (Fig. 1). After 1 week, the fibers were markedly reduced in size and separated by interstitial edema which was more evident in the perimysiuni (Fig. lb). T h e fibers displayed focal niyofibrillar disruption, but histochemical ATPase activity appeared normal, as previously reported.2222 Sparse fibers showed a coarse reaction for NADH-d activity. After a 2-week suspension, muscle fascicles Morphological Observations.
RESULTS
In suspension, group A rabbits showed, most of the time, characteristic postural changes with their hindfeet plantarflexed up to 180", but they occasionally fully extended and flexed their liindlinibs.2,22Group R rabbits, suspended with the hind feet dorsiflexed, General Observations.
Table 1. Quantitative data
1 week
Group
Body weight (9)
Soleus wet weight (rng)
A B
1200 (?66)* 1214 (229)* 1303 (233) 1258 ( t 4 2 ) * 1284 (239)* 1541 ( t 4 5 ) 1439 (? 75)*
555 (*38)* 649 ( 232) 682 (129) 430 (k32)* 772 (123) 812 (144) 633 (k60)'
L
2 weeks
A
4 weeks
B C A B C
-
2199 (2179)
Soleus to body weight ratio ( x 0.45 (+0.008)* 0.52 (20.01) 0.52 (kO.01) 0.33 (+0.01)* 0.59 (20.005)* 0.52 (50.01) 0.43 (?0.01)*
Soleus cross fiber area (Km') 931 (+46)* 1228 (259) 1254 (253) 492 (2238)" 1252 (2244) 1291 ( t 1 2 8 ) -
-
-
-
1108 (256)
0.49 (20.01)
-
A = hindlimb suspension, B = hindlimb suspension with foot dorsiflexion C = control Values are means -c SD (n = 5) *Significant vs matched C group with P 5 0 01 using the one way ANOVA test
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FIGURE 1. Cryostat sections of rabbit Soleus muscles stained for modified Gomori's trichrome (a-e) or NADH-d activity (f). Compared to control soleus (a), progressive muscle atrophy develops at 1 (b), 2 (c, e, and f ) , and 4(d) weeks HS. Two branched fibers (arrows) and central myonuclei are evident in (e). NADH-d activity (f) appears to be high and coarsely distributed in most of the fibers, sometimes with prominent subsarcolemmal staining. Interstitial edema is more evident at 1 week and adipose tissue at 4-week-HS. (a,b,c,d: bar = 33 pm; e,f: bar = 13 km).
were markedly reduced in size, while the perimysium appeared focally enlarged and occupied by adipose tissue (Fig. Ic). Reactive cells had extensively infiltrated the endomysium, and single
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erythrocytes were sometimes observed free in the connective tissue (Fig. 2). All muscle fibers were atrophic, round, or polygonal in shape (Figs. lc and 2). T h e myofibrillar
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FIGURE 2. Semithin cross (a) and longitudinal (b) sections of Soleus muscle fibers after 2-week suspension. The fibers demonstrate an irregular outline of sarcolemma and myofibrils are no longer recognizable. Monocytic cells infiltrate the endomysium. Rare extravascular erythrocytes are clearly discernible (bar = 10 prn).
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FIGURE 3. Electron micrographs of rabbit soleus fibers after 2-week suspension. (a) Myofibrils are no longer recognizable (bar = 1 pm). (b) Parts of two atrophied muscle fibers, showing an indented profile (bar = 2 pm). (c) Atrophied muscle fiber covered with redundant folds of basal lamina, containing dotlike densities of uncertain origin (bar = 0.25 pm).
ATPase reaction was generally lower than normal, and the partial lack of staining in some fibers indicated regional myofibrillar loss. T h e NADH-d reaction was coarse in most fibers suggesting a wide-
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spread disruption of the intermyofibrillar network (Fig. I f ) . Ultrastructurally, very limited fiber regions retained short disaligned sarcomeres. In general,
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the sarcomeric pattern was completely lost and the myofibrillar components were disorganized and aberrant (Figs. 2b and 3a). Z-lines were fragmented and pulled apart. A- and I-bands were no longer recognizable during this portion of the experiment, appearing as remnants of disarranged filaments. Triads were unassociated with myofila-
ments, and mitochondria were small and electrondense, isolated, or grouped in clusters (Figs. 3a,b and 4);ribosomes were sparse and glycogen granules were rare. In some fibers, among areas of myofibrillar breakdown, reactive sarcoplasmic organelles made their appearance, such as abundant Golgi apparatus, T-system profiles, and proliferat-
FIGURE 4. Electron micrograph of rabbit soleus after 2-week suspension. A myelinated axon innervates an atrophic muscle fiber devoid of well-preserved myofibrils. Axon terminals contain swollen mitochondria, while the second terminal from the left is fragmented. The capillary on the top shows abnormal endothelial cell processes. Numerous euchromatic nuclei and hyperdense mitochondria are present in the postsynaptic region (bar = 2 Fm).
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ing cisternae of sarcoplasmic reticulum. Nuclei were round or lobulated with sparse chromatin; they were generally located in the subsarcolemmal region (Figs. le, 2, and 3b). Atrophic myofibers displayed an indented profile (Maine coastline type) and the sarcolemma showed a rich pinocytotic inpocketing (Figs. 2 and 3b,c). Although the basal lamina followed the general outline of the muscle fibers, it was redundant, forming fingerlike projections on the cell surface (Fig. 3c). Rows of granular densities were generally entrapped inside the fingerlike projections of the basal lamina (Fig. 3c). Altogether, these changes appeared to indicate acute severe volumetric reduction of the muscle fibers. Regenerating muscle cells were also identified (2% to 4%) histologically as branched or intensely basophilic cells, with large central nuclei at this stage of the experiment (Fig. le). Ultrastructurally, they appeared at different stages of muscle differentiation (Fig. 5). Some were small, round cells with central euchromatic nucleus, abundant sarcoplasm, sparse mitochondria, and rare myofibrils. These cells were provided with short stretches of basal lamina. Others had the appearance of more mature muscle cells, with a peripheral nucleus, abundant myofibrils, small collections of mitochondria, and a continuous basal lamina. Unlike the atrophic fibers described above, regenerating fibers had smooth cell profiles, and the basal lamina was tightly fitted. Finally, only 0.5% to 1% of the fibers in the midbelly region appeared necrotic: sarcoplasmic organelles were no longer recognizable, and were replaced by granular debris, filamentous fragments, and autophagic vacuoles. The plasma membrane was lost in several points. After 4-week suspension, the structure of the soleus muscle was completely subverted (Fig. Id). The architecture of the perimysium was no longer recognizable, replaced by large and irregular areas of fat tissue as demonstrated by trichrorne and Oil Red 0 staining. The muscle fascicles appeared sparse and were separated from each other by irregular areas of adipose tissue. It was noteworthy that the interstitial edema as well as mononucleated reactive cells, that were infiltrating the periand the endomysium at 2 weeks, were not observed at this stage of the experiment. Muscle fibers were generally atrophic and their cross-sectional area was highly variable (Fig. Id). Muscle fibers displayed scalloped profile and leftover scanty disarrayed myofibrils (Fig. 6a,b). Remnant muscle fibers were reduced to a collection of
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myonuclei (nuclear clumps or chains), surrounded by a narrow rim of cytoplasm devoid of myofibrils. However, a few, sparse, minute fibers had a virtually normal sarcomeric pattern (2% to 3%) (Fig. 6b). Their outline was either smooth and round, or scalloped and irregular with fingerlike projections. Large blood vessels and nerve fascicles were unremarkable throughout the experiment. However, after 2- and 4-week suspensions, 2 of 14 presynaptic terminals were fragmented and 8 of 14 showed vacuolated mitochondria (Fig. 4). Capillaries sported abnormal endothelial cell processes projecting into the vessel lumen (Fig. 4). Muscle spindles were grossly normal throughout the experiment, as seen in light microscopy. Morphologzc Evaluation of Group B Animals. For the first 2 weeks of suspension, the soleus muscle fibers of this experimental group, examined in cross sections, were indistinguishable from those of the control group. When examined in the longitudinal section, myofibers showed a regular outline and the sarcomeric pattern appeared to be preserved (Fig. 7). Morphometric Data. The mean cross-fiber area in the soleus of group A rabbits was 25% and 6 1.6% less than that of control rabbits after 1 and 2 weeks, respectively. On the other hand, the cross-fiber area of group B animals was similar to that of control animals at both time intervals (Table 1). DISCUSSION
Two conclusions to be drawn from this study are that HS causes a progressive myofiber damage in rabbit soleus, and that maintenance of passive tension and proper length prevents that damage in the unloaded soleus. The pathological process is not selective for a soleus fiber type. It may be divided into three characteristic stages, observable respectively after 1-, 2-, and 4-week suspensions. Multifocal myofibrillar disruption occurs at stage I. Stage I1 is characterized by myofiber atrophy, widespread myofibrillar damage, and limited muscle regeneration. Muscle atrophy and substitution by adipose tissue are the main features of stage 111. Obviously, these are not three discontinuous stages: one follows the other in a continuous fashion, with overlapping between the stages and carryover of certain features from one stage to the next. As previously reported, in stage I, muscle damage is multifocal and extended across the width of the fibers. It consists of myofilament derange-
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FIGURE 5. Rabbit soleus after 2-week suspension. A fully regenerated but minute muscle fiber displays straight plasmalemma and basement membrane (upper right), while a regenerating muscle cell has an incomplete basal lamina covering (bottom left) (bar = 1 Pm).
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FIGURE 6. Rabbit soleus after 4-week suspension. (a) Semithin cross section. Surviving muscle fibers are small in size and display a jagged profile, while numerous blobs of fat infiltrate the endo- and perimysial space (bar = 40 pm). (b) Semithin longitudinal section. A lean muscle fiber with a villous profile shows almost regular sarcomeric pattern, while other fibers appear to be reduced to linear collections of myonuclei (bar = 40 pm).
Reduced Length Damages Soleus
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FIGURE 7. Rabbit soleus after 2-week suspension with the feet dorsiflexed. Semithin sections. (a) A muscle fascicle with polygonal fibers and normal endomysial space. (b) Myofibrils have a normal sarcomeric pattern (bar = 10 pm).
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ment associated with loss of Z-bands and mitotral core formations in the tenotomized rat soleus chondria.2922 seems to be part of the reparative process reconIn stage 11, muscle damage is different in that stituting the continuity of the sarcomeric banding it affects all components of the myofibers and it is pattern until normal fiber histology is restored.3 widespread rather than focal. Sarcomeres are vanFinally, rare fibers adapt to HS in rabbit soleus, ishing and the whole myofiber loses myofibrillar showing normal myofilament overlap and proper and sarcoplasmic fractions. Atrophy proceeds sarcomeric length, while the soleus remains at a faster in the fiber inner compartment than in the substantially shortened length. Why significant replasmalemma. parative changes are not expressed or are inhibIf HS persists for 4 weeks, atrophy of the soited in the unloaded rabbit soleus is unexplained. leus progresses to an extreme state in which part A species-specific characteristic can be advocated of the fibers are reduced to nuclear chains proin considering the sensitivity of rabbit soleus to vided with undifferentiated sarcoplasm. 'These unloaded shortening. cells share features with the so-called s a r c ~ c y t e s . ~ ~ Although muscle regeneration clearly develops This complex process does not seem to be susafter the second week of HS, the regenerative tained by lysosomal proteolysis in rabbit, because process does not counterbalance the progressive autophagic vacuoles were not observed in atrophic destruction of myofibrils, because regenerated fifibers. Fast atrophy of the soleus during HS has bers are still small and few at stage 111. The lack been suggested to be secondary to increased of' passive stretch during HS affects probably also ubiquitin-dependent protein degradation in rat." the growth of' regenerating fibers. On the other hand, the wasted muscle tissue is reChronic blood congestion obviously occurs in placed mainly by proliferated adipocytes, accountthe venous system of suspended hindlimbs.20 The ing for the increase in mass of the suspended sopossibility that compromised blood flow, demonstrated in suspended hindlimbs,14 affects the releus between 2- and 4-week suspensions. parative process of damaged soleus fibers, cannot The microscopic changes show evidence that be ruled out with certainty. the loss of sarcomeres in the soleus progresses to The morphological changes observed in axon involve the entire observable course of the fibers, terminals of suspended rabbit soleus appear to be probably beyond the limit required for the fibers mild and evident when soleus atrophy is already to adjust to the shorter length determined by HS.2 marked. They are probably retrograde in type or In the atrophic soleus fibers of suspended rabbit secondary to the severe atrophy of the dependent there are no significant signs of a reactive commuscle fibers. In the unloaded rat soleus, neuropensatory process. The nuclei appear quiescent, muscular junctions have been reported to be norlocalized in the subsarcolemmal region in most of ma1.20 the fibers, while ribosomes and sarcoplasmic reticUnlike the soleus, the medial and lateral porulum are scanty. 'Therefore, indirect signs of intion of gastrocnemius show only moderate atrocreased protein synthesis in atrophic fibers are phy affecting both type I and type I1 fibers with lacking. no alteration of the histoenzymatic patterns, even The similarities between muscle changes deterafter 4-week HS (unpublished observations). mined by HS or by tenotomy in rabbit soleus are noteworthy because tenotomized muscle displays Effects of Dorsiflexion. Avoiding hindlimb posmyofibrillar derangement, severe atrophy, and fat tural changes in the suspended rabbit prevents sosubstitution after several The two exleus muscle damage. It is surprising that dorsiflexperimental conditions determine a comparable bioion of the feet and sporadic contractions against mechanical change for the soleus consisting o f elastic resistance effectively prevent, not only myoseverely reduced working length and phasic confibrillar breakdown, but also muscle atrophy in traction without a load.'" unloaded hindlimbs. Stretching the hypoactive soContrary to that observed in rabbit, a better leus fibers is probably suf-ficient, at least for a few adaptation to sustained shortening, secondary to weeks, in preserving the balance between protein immobilization or tenotomy, takes place in rat and synthesis and degradation, despite the lack of cat soleus."" It first comprises a decrease in the load-bearing activities. number of sarcomeres in the midbelly region and, The type of foot dorsiflexion we used seems subsequently, a fast reorganization of myofibrillar pertinent to the specific biomechanical conditions apparatus, eventually attaining optimal myofiladetermined both by HS and hypogravity, in which ment o~erlap."'~The typical appearance of cen-
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there is a lack of resistance to the contraction of antigravity muscles.22 By applying an elastic resistance to shortening of antigravity muscle during HS, hindlimbs were neither immobilized nor engaged in supporting body wei ht as is the case in other experimental models. 10f1313 In our experimental approach, namely in group B rabbits, the specific working length of the soleus is, to some extent, respected during HS: in the resting posture, the soleus is fully stretched, while it becomes shortened against resistance during the plantar extension of the hindfeet. Such a simple device can be easily adapted to the feet of astronauts. It would have a preventive effect also on hypogravityinduced soleus muscle atrophy. 12s19-21 T h e new insight resulting from the above discussion is that an hypothesis can be formulated regarding the sensitivity of the soleus to HS. It is a tonic antigravity muscle, composed chiefly or almost exclusively of type I fibers, provided with distinctive biochemical and ultrastructural features such as elevated calcium exchange and characteristically wide Z-lines.",' Physiologically, during quadrupedal standing, the soleus muscle is stretched and, at the same time, it appears to be close to maximal and virtually continuous activity, whereas it is inactive and electrically silent during the nonstance phase of a ~ t e p . ' , ~Thus, ~ ' ~ ,tonic ~~ muscle contractions of the soleus are, in general, isometric rather than isotonic. This implies that soleus muscle myofibrils usually develop maximum contractile activities with little or no sliding of myofilaments. Conversely, by removing the constant tension imposed by load-bearing activity, isotonic contraction may occur in the soleus, but without important transfer of energy. Electromyographic recordings in rats support the hypothesis that an active shortening may take place in suspended soleus. Thus, soleus myofibrils can develop tonic and phasic contractions in an abnormally shortened state, compromising the intermyofilament bridges and the architecture of myofilament overlap and of the myofiber cytoskeleton. The shorter is the soleus and the longer its permanence in such a state, the greater the myofibrillar damage. In conclusion, isometric muscle contraction represents the optimal working condition for the soleus and, at the same time, may guarantee maintenance of its integrity. When this condition is subverted, tension-related myofibrillar damage can be expected. This may apply to a variety of pathologic conditions involving the activation of the fibers either in an abnormally shortened or
'.*'
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lengthened state, such as hypogravity, tenotomy, immobilization, spasticity due to CNS disorders, and lengthening contractions .3,722,1 871972 53
'
'
REFERENCES 1. Alford EK, Roy RR, Hodgson JA, Edgerton VR: Electro-
of rat soleus, medial gastrocnemius, and tibialis anterior during hind limb suspension. Exp Neurol 1987;96:635-649. 2. Anzil AP, Sancesario G, Massa R, Bernardi G: Myofibrillar disruption in the rabbit soleus muscle after one-week hindlimb suspension. Muscle Nerve 1991;14:358-369. 3. Baker JH: Segmental necrosis in tenotomized muscle fibers. Muscle Nerve 1983;6:29-39. 4. Bergman RA, Afifi AK: T h e structure of the rabbit soleus muscle and the structural alterations resulting from tenotomy.John Hopkinr MedJ 1969;124:119-131. 5 Corley K, Kovalchuk N , McComas AJ: Contrasting effects of suspension on hind limb muscles in the hamster. Exp Neurol 1984;85:30-40. 6 Everts ME, Clausen T : Effects of thyroid hormones on calcium contents and 45Ca exchange in rat skeletal muscle. Am J Physiol 1986;251:E258-E265. 7 Ferguson A, Vaugan L, Ward L: A study of disuse atrophy of skeletal muscle in the rabbit. J Bone Joint Surg 1957;39:583- 596. 8. Gardiner KF, Gardiner PF, Edgerton VR: Guinea pig soleus and gastrocnemius electromyograms at varying speeds, grades and loads. J Appl Physiol 1982;52:451-457. 9. Gauthier GF: Some ultrastructural and cytochemical features of fiber populations in the soleus muscle. Anat Rec 1975; 18O:551-564. 10. Graham SC, Roy RR, West SP, Thomason D, Baldwin KM: Exercise effects on the size and metabolic properties of soleus fibers in hindlimb-suspended rats. Auial Space Environ Med 1989;60:226-234. 11. Herbert ME, Roy RR, Edgerton VR: Influence of oneweek hindlimb suspension and intermittent high load exercise on rat muscles. Exp Neurol 1988;102:190- 198. 12. Ilyina-Kakueva EI, Portugalov VV, Krivenkova P: Space Hight effects on the skeletal muscles of rats. Aviat Space Enuiron Med 1976;47:700-703. 13. Jaspers SR, Fagan JM, Satarug S, Cook PH, Tischerl ME: Effects of immobilization on rat hind limb muscles under non-weight-bearing conditions. Muscle Nerve 1988; 11:458466. 14. LeBlanc A , Marsh C;, Evans H, Johnson P, Schneider V, Jhingran S: Bone and muscle atrophy with suspension of the rat. J Appl Physiol 1985;58:1669- 1675. 15 McMinn RM, Vrbova G: Morphological changes in red and pale muscles following tenotomy. Nature 1962; 195:509. 16 McMinn RM, Vrbova G: Motoneurone activity as a cause of degeneration in the soleus muscle of the rabbit. Q J Exp Physiol 1967;52:4 1 1- 4 15. 17 Musacchia XJ, Deavers DR, Meininger GA, Davis TP: A model for hypokinesia: Effects on muscle atrophy in the rat. J Appl Physiol 1980;48:479-486. 18 Ogilvie RV, Armstrong RB, Baird KE, Bottoms CL: Lesion in the rat soleus muscle following eccentrically biased exercise. Am J Anat 1988; 182:335-346. 19. Riley DA, Ellis S, Slocum GK, Satyanarayana T, Bain JLW, Sedlak FR: Hypogravity-induced atrophy of rat soleus and extensor digitorum longus muscles. Muscle Nerue 1987; 1 O:56O- 568. 20. Riley DA, Slocum GR, Bain JLW, Sedlak FR, Sowa TE, Mellender J W: Rat hindlimb unloading: soleus histochemImyography
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istry, ultrastructure, and electromyography. J Appl Physiol 1990;69:58-66. Riley DA, Ilyina-Kakueva EI, Ellis S, Bain JLW, Slocum GR, Sedlak FR: Skeletal muscle fiber, nerve, and blood vessel breakdown in space-flown rats. FASEBJ 1990;4:84-91. Sancesario G, Massa R, Anzil AP, Bernardi G : Hindlimb suspension of rabbit for one week: myofibrillar damage in the soleus may depend on long-lasting shortening and periodic contraction against no load, in Proceedings of the Fourth European Symposium on Life Science Research in Space (ESA SP-307), 1990, pp 375-380. Smith JL, Edgerton VR, Betts B, Collatos TC: EMG of slow and fast ankle extensors of cat during posture, locomotion and jumping. J Neurophysiol 1977;40:503-513. Steffen JM, Robb R, Dombrowski MJ, Musacchia J X , Man-
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del AD, Sonnenfeld G: A suspension model for hypokinetic-hypodinamic and antiorthostatic responses in the mouse. Aviat Space Enuiron Med 1984;55:612-616. 25. Tabary JC, Tabary C, Tardieu C, Tardieu G, Goldspink G: Physiological and structural changes in the cat’s soleus muscle due to immobilization at different lengths by plaster cdsts. J Physiol 1972;224:231-244. 26. Templeton GH, Padalino M, Manton J, Glasberg M, Silver CJ, Silver P, DeMartino G , Leconey T, Klug G, Hagler H, Sutko JL: Influence of suspension hypokinesia on rat soleus muscle. J Appl Physiol 194:;56:278-286. 27. Wohlfart G , Henriksson K: General pathology of skeletal muscle, in Haymaker W, Adams RD (eds): Histology and Histopathology of the Nervous System, Springfield, 1L: Charles Thomas, 1984, vol 11, pp 1690- 1734.
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ACTIVE MUSCLE LENGTH REDUCTION PROGRESSIVELY DAMAGES SOLEUS I N HINDLIMB-SUSPENDED RABBITS GIUSEPPE SANCESARIO, MD, ROBERTO MASSA, MD, ARCHINTO P. ANZIL, MD, and GlORGlO BERNARDI, MD
Hindlimb suspension techniques have been increasingly used as a simple and inexpensive ground-based model in order to reproduce some effects of weightlessness on antigravity muscles of small mammals. HS in experimental animals as well as microgravity in astronauts eliminate hindlimb-supported body weight. It seems obvious that antigravity muscles produce little or no force under these conditions. ' 2 , L 7 Unexpectedly, chronically recorded electromyograms in rats show almost normal levels of electrical activity in the unloaded muscles, particularly in the tibialis anterior, the soleus, and the gastrocnemius, during From the Laboratory of Experimental Neuropathology. Institute of Neurology, Second University of Rome, Italy (Drs Sancesario, Massa. and Bernardi); and the Department of Pathology, SUNY-HSCE, Brooklyn, New York (Dr. Anzil). Acknowledgments. This study was supported in part by a grant from the Agenzia Spaziale ltaliana (AS1 90-RS-30). Address reprint requests to Dr Giuseppe Sancesario, Laboratory 01 Experimental Neuropathology, Institute of Neurology, Second University of Rome, Via 0.Raimondo 8, 00173 Rome, Italy. Accepted lor publication February 3, 1992 CCC 0148-639X1921091002-14 $04.00 0 1992 John Wiley & Sons, Inc.
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'
prolonged HS. Moreover, the contractile activity of the soleus, a tonic muscle, shifts to a phasic pattern after the fourth day of HS." In spite of persistent muscle activity, up to 50% reduction in muscle weight arid pertinent morphologic changes have been reported to develop in the soleus of the rat, mouse, and hamster after the first week of ~ ~ . 5 , 1 7 , 2Although 1 conditions of hypokinesiahypodynamia obviously affect suspended hindlimbs,"*26 the mechanism by which unloading of the hindlimbs specifically damages the soleus fibers is not clear. In recent experiments, we have observed that HS of rabbits for 1 week produces, in soleus myofibers, multifocal areas of myofibrillar disruption concomitant with muscle atrophy.* T h e myofibrillar- lesions were thought to be dependent on both the conditions of chronic shortening and periodic contraction against no load occurring in suspended soleus.27'L2Similar conclusions have recently been drawn from experiments in HS of rats. 'Lo With this in mind we can hypothesize that segmental loss of sarcomeres in the soleus during the first week of HS may represent a cytoarchitectural
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remodeling of the myofibers in an attempt to adjust to their new and shorter working length.3 Alternatively, we can presume that phasic and tonic soleus contractions without a load, if they are long-lasting, may constitute a persistent detrimental factor, leading to widespread muscle damage. Either way, the abnormal shortening of the soleus would play a major part in causing myofibrillar changes. The present study was designed to investigate whether the segmental myofibrillar damage induced by HS in the soleus is a self-limiting or a worsening process, roughly proportional to the duration of suspension. In addition, we attempted to prevent the occurrence of myofibrillar disruption in the soleus by avoiding the reduction of muscle length during HS. Our approach was to apply an elastic resistance to the plantar flexion of the feet of suspended rabbits. In this way, the unloaded hindlimbs could be actively extended against resistance, while postural changes determined by suspension were prevented. Data are presented on the severe damage to myofibers determined in the soleus muscle by prolonged HS. Furthermore, we report here on the finding that the resistance imposed on chronic shortening and on phasic soleus muscle contractions is essential in maintaining mass and structural fiber integrity during unloading of the hindlimbs. Appropriate muscle length and resistance to contraction may represent the specific biomechanical requirements for the physiology of the antigravity soleus muscle, engaged mainly in developing long-lasting isometric contractions. ANIMALS AND METHODS
The experiments were performed on young, male New Zealand rabbits (1025 to 1150 g body weight), randomly assigned to three groups: ground-based control (group C, n = 24), hindlimb suspended (group A, n = 24), and hindlimb suspended plus passive dorsiflexion of the feet (group B, n = 16). Animal experimentation was conducted in accordance with the institution's guide for the care and use of laboratory animals. Control rabbits were kept in standard laboratory conditions. Experimental rabbits were suspended in a horizontal position as previously reported.2z22In brief, the animals were dressed with a denim harness and were suspended from a hook so that, while forelimbs stood on a wooden support, hindlimbs dangled down freely for 1, 2, or 4 weeks. Animals in group B were also suspended
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according to this model for 1 or 2 weeks; however, a dorsiflexed posture of the feet at 45" was passively maintained with a rubber band fastened to the foot at one end and to the denim harness at the other end. The length of the rubber band and its fastening to the harness were selected so that unloaded hindlimbs were kept flexed as in the control animals at rest on the floor of the cage. The resistance of the rubber band was empirically chosen so that animals could extend their hindlimbs without moving their body wrapped in the harness. In stretching the rubber band by 6 cm, the ankle joint angle could be opened from 45" to go", which is the estimated range of ankle movement in walking rabbits before HS. A weight of 270 to 280 g, comparable to 21% to 26% of the body weight of suspended animals, was evaluated to be sufficient to stretch an extremity of the rubber band by 6 cm. Body weight was assessed before and after the period of suspension. Food intake was measured daily. The behavior of the suspended animals was also observed daily for 30 minutes in the morning and 30 minutes in the afternoon. At the end of a given suspension period, the animals were immediately sacrificed under barbiturate/ketamine anesthesia; care was taken to avoid reloading the hindlimbs in removing the animals from the suspension apparatus. A t each stage of the experiment 3 animals from each group were used. The solei were excised, the midbelly regions were mounted in tragacanth gum and quickly frozen in isopentane cooled in liquid nitrogen. Cryostat sections (10 Fm) were stained either for modified Gomori's trichrome, alkaline and acid myofibrillar adenosinetriphosphatase (ATPase), nicotinamide adenine dinucleotide dehydrogenase (NADH-d), or for Oil Red 0. The type of fibers, and the numbers of necrotic or regenerating ones, were evaluated in control and suspended animals in at least 200 fibers from randomly selected fields of each muscle in pertinent histological and histochemical sections. Histochemistry.
Five animals from each group were used at each stage of the experiment. The hindquarters were perfused through the abdominal aorta with a 4% glutaraldehyde solution in 0.1 mol/L phosphate buffer, pH 7.4. After perfusion, the soleus muscles were removed, weighed, and kept in cold perfusate overnight. Under a stereomicroscope several samples, ap-
Electron Microscopy.
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also occasionally extended their hindlimbs u p to 100" against the resistance created by the rubber band. Food intake was about 20% less in groups A and B animals for a few days; afterward, it was the same as for group C animals. However, the gain in body weight was significantly reduced after the first week of suspension in groups A and B (Table 1). The wet weight of the soleus in group A animals was 18.6%, 47.6%, and 41.9% less than in control animals after 1, 2, and 4 weeks, respectively. On the contrary, in group €3 animals, the wet weight of the soleus was not significantly different from that of control animals after 1 and 2 weeks, respectively (Table 1). Compared with control animals, the soleus weighdbody weight ratio was significantly lower in group A, whereas, in group €3, it was equal at 1-week and even higher after 2-week suspension.
proximately 2 x 2 mm, were cut from the midbelly region of the muscle. T h e pieces were rinsed in buffer, postfixed for 90 minutes in 1% buffered osmium tetroxide (at 20" C) and conventionally embedded in Epon 812. Semithin sections were cut according to a transverse or longitudinal plane with an LKB ultramicrotome and stained with paraphenylenediamine. They were viewed and photographed with a phase contrast Leitz automatic photomicroscope. Thin (silver) sections were stained with uranyl acetate and lead citrate and examined with a Zeiss EM-10 electron microscope. T h e number of fibers with a recognizable sarcomeric banding pattern was evaluated in random fields after scoring an average of 250 fibers per muscle in longitudinal semithin sections. T h e morphometric evaluation was made using a MOP-3 image analysis system (Zeiss) on photographic prints of muscle semithin transverse sections. 'The mean cross fiber area was calculated in random fields of soleus from both sides of each animal, after measuring an average of 250 30 fibers per muscle. Because of the clear-cut difference between the two experimental groups, the comparison was limited to the results obtained at 1- and 2-week suspension. Soleus weight to body weight ratios and measurements of cross fiber areas were analyzed using the one-way ANOVA test.
*
Muscle fibers with moderately high NADH-d activity predominated in the control rabbit soleus. Fibers staining dark with acid ATPase reaction represented 80% to 85% of' the fiber population. Group A Animal.\. 'Ihe longer the period of suspension, the greater the changes observed in soleus fibers (Fig. 1). After 1 week, the fibers were markedly reduced in size and separated by interstitial edema which was more evident in the perimysiuni (Fig. lb). T h e fibers displayed focal niyofibrillar disruption, but histochemical ATPase activity appeared normal, as previously reported.2222 Sparse fibers showed a coarse reaction for NADH-d activity. After a 2-week suspension, muscle fascicles Morphological Observations.
RESULTS
In suspension, group A rabbits showed, most of the time, characteristic postural changes with their hindfeet plantarflexed up to 180", but they occasionally fully extended and flexed their liindlinibs.2,22Group R rabbits, suspended with the hind feet dorsiflexed, General Observations.
Table 1. Quantitative data
1 week
Group
Body weight (9)
Soleus wet weight (rng)
A B
1200 (?66)* 1214 (229)* 1303 (233) 1258 ( t 4 2 ) * 1284 (239)* 1541 ( t 4 5 ) 1439 (? 75)*
555 (*38)* 649 ( 232) 682 (129) 430 (k32)* 772 (123) 812 (144) 633 (k60)'
L
2 weeks
A
4 weeks
B C A B C
-
2199 (2179)
Soleus to body weight ratio ( x 0.45 (+0.008)* 0.52 (20.01) 0.52 (kO.01) 0.33 (+0.01)* 0.59 (20.005)* 0.52 (50.01) 0.43 (?0.01)*
Soleus cross fiber area (Km') 931 (+46)* 1228 (259) 1254 (253) 492 (2238)" 1252 (2244) 1291 ( t 1 2 8 ) -
-
-
-
1108 (256)
0.49 (20.01)
-
A = hindlimb suspension, B = hindlimb suspension with foot dorsiflexion C = control Values are means -c SD (n = 5) *Significant vs matched C group with P 5 0 01 using the one way ANOVA test
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FIGURE 1. Cryostat sections of rabbit Soleus muscles stained for modified Gomori's trichrome (a-e) or NADH-d activity (f). Compared to control soleus (a), progressive muscle atrophy develops at 1 (b), 2 (c, e, and f ) , and 4(d) weeks HS. Two branched fibers (arrows) and central myonuclei are evident in (e). NADH-d activity (f) appears to be high and coarsely distributed in most of the fibers, sometimes with prominent subsarcolemmal staining. Interstitial edema is more evident at 1 week and adipose tissue at 4-week-HS. (a,b,c,d: bar = 33 pm; e,f: bar = 13 km).
were markedly reduced in size, while the perimysium appeared focally enlarged and occupied by adipose tissue (Fig. Ic). Reactive cells had extensively infiltrated the endomysium, and single
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erythrocytes were sometimes observed free in the connective tissue (Fig. 2). All muscle fibers were atrophic, round, or polygonal in shape (Figs. lc and 2). T h e myofibrillar
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FIGURE 2. Semithin cross (a) and longitudinal (b) sections of Soleus muscle fibers after 2-week suspension. The fibers demonstrate an irregular outline of sarcolemma and myofibrils are no longer recognizable. Monocytic cells infiltrate the endomysium. Rare extravascular erythrocytes are clearly discernible (bar = 10 prn).
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FIGURE 3. Electron micrographs of rabbit soleus fibers after 2-week suspension. (a) Myofibrils are no longer recognizable (bar = 1 pm). (b) Parts of two atrophied muscle fibers, showing an indented profile (bar = 2 pm). (c) Atrophied muscle fiber covered with redundant folds of basal lamina, containing dotlike densities of uncertain origin (bar = 0.25 pm).
ATPase reaction was generally lower than normal, and the partial lack of staining in some fibers indicated regional myofibrillar loss. T h e NADH-d reaction was coarse in most fibers suggesting a wide-
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spread disruption of the intermyofibrillar network (Fig. I f ) . Ultrastructurally, very limited fiber regions retained short disaligned sarcomeres. In general,
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the sarcomeric pattern was completely lost and the myofibrillar components were disorganized and aberrant (Figs. 2b and 3a). Z-lines were fragmented and pulled apart. A- and I-bands were no longer recognizable during this portion of the experiment, appearing as remnants of disarranged filaments. Triads were unassociated with myofila-
ments, and mitochondria were small and electrondense, isolated, or grouped in clusters (Figs. 3a,b and 4);ribosomes were sparse and glycogen granules were rare. In some fibers, among areas of myofibrillar breakdown, reactive sarcoplasmic organelles made their appearance, such as abundant Golgi apparatus, T-system profiles, and proliferat-
FIGURE 4. Electron micrograph of rabbit soleus after 2-week suspension. A myelinated axon innervates an atrophic muscle fiber devoid of well-preserved myofibrils. Axon terminals contain swollen mitochondria, while the second terminal from the left is fragmented. The capillary on the top shows abnormal endothelial cell processes. Numerous euchromatic nuclei and hyperdense mitochondria are present in the postsynaptic region (bar = 2 Fm).
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ing cisternae of sarcoplasmic reticulum. Nuclei were round or lobulated with sparse chromatin; they were generally located in the subsarcolemmal region (Figs. le, 2, and 3b). Atrophic myofibers displayed an indented profile (Maine coastline type) and the sarcolemma showed a rich pinocytotic inpocketing (Figs. 2 and 3b,c). Although the basal lamina followed the general outline of the muscle fibers, it was redundant, forming fingerlike projections on the cell surface (Fig. 3c). Rows of granular densities were generally entrapped inside the fingerlike projections of the basal lamina (Fig. 3c). Altogether, these changes appeared to indicate acute severe volumetric reduction of the muscle fibers. Regenerating muscle cells were also identified (2% to 4%) histologically as branched or intensely basophilic cells, with large central nuclei at this stage of the experiment (Fig. le). Ultrastructurally, they appeared at different stages of muscle differentiation (Fig. 5). Some were small, round cells with central euchromatic nucleus, abundant sarcoplasm, sparse mitochondria, and rare myofibrils. These cells were provided with short stretches of basal lamina. Others had the appearance of more mature muscle cells, with a peripheral nucleus, abundant myofibrils, small collections of mitochondria, and a continuous basal lamina. Unlike the atrophic fibers described above, regenerating fibers had smooth cell profiles, and the basal lamina was tightly fitted. Finally, only 0.5% to 1% of the fibers in the midbelly region appeared necrotic: sarcoplasmic organelles were no longer recognizable, and were replaced by granular debris, filamentous fragments, and autophagic vacuoles. The plasma membrane was lost in several points. After 4-week suspension, the structure of the soleus muscle was completely subverted (Fig. Id). The architecture of the perimysium was no longer recognizable, replaced by large and irregular areas of fat tissue as demonstrated by trichrorne and Oil Red 0 staining. The muscle fascicles appeared sparse and were separated from each other by irregular areas of adipose tissue. It was noteworthy that the interstitial edema as well as mononucleated reactive cells, that were infiltrating the periand the endomysium at 2 weeks, were not observed at this stage of the experiment. Muscle fibers were generally atrophic and their cross-sectional area was highly variable (Fig. Id). Muscle fibers displayed scalloped profile and leftover scanty disarrayed myofibrils (Fig. 6a,b). Remnant muscle fibers were reduced to a collection of
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myonuclei (nuclear clumps or chains), surrounded by a narrow rim of cytoplasm devoid of myofibrils. However, a few, sparse, minute fibers had a virtually normal sarcomeric pattern (2% to 3%) (Fig. 6b). Their outline was either smooth and round, or scalloped and irregular with fingerlike projections. Large blood vessels and nerve fascicles were unremarkable throughout the experiment. However, after 2- and 4-week suspensions, 2 of 14 presynaptic terminals were fragmented and 8 of 14 showed vacuolated mitochondria (Fig. 4). Capillaries sported abnormal endothelial cell processes projecting into the vessel lumen (Fig. 4). Muscle spindles were grossly normal throughout the experiment, as seen in light microscopy. Morphologzc Evaluation of Group B Animals. For the first 2 weeks of suspension, the soleus muscle fibers of this experimental group, examined in cross sections, were indistinguishable from those of the control group. When examined in the longitudinal section, myofibers showed a regular outline and the sarcomeric pattern appeared to be preserved (Fig. 7). Morphometric Data. The mean cross-fiber area in the soleus of group A rabbits was 25% and 6 1.6% less than that of control rabbits after 1 and 2 weeks, respectively. On the other hand, the cross-fiber area of group B animals was similar to that of control animals at both time intervals (Table 1). DISCUSSION
Two conclusions to be drawn from this study are that HS causes a progressive myofiber damage in rabbit soleus, and that maintenance of passive tension and proper length prevents that damage in the unloaded soleus. The pathological process is not selective for a soleus fiber type. It may be divided into three characteristic stages, observable respectively after 1-, 2-, and 4-week suspensions. Multifocal myofibrillar disruption occurs at stage I. Stage I1 is characterized by myofiber atrophy, widespread myofibrillar damage, and limited muscle regeneration. Muscle atrophy and substitution by adipose tissue are the main features of stage 111. Obviously, these are not three discontinuous stages: one follows the other in a continuous fashion, with overlapping between the stages and carryover of certain features from one stage to the next. As previously reported, in stage I, muscle damage is multifocal and extended across the width of the fibers. It consists of myofilament derange-
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FIGURE 5. Rabbit soleus after 2-week suspension. A fully regenerated but minute muscle fiber displays straight plasmalemma and basement membrane (upper right), while a regenerating muscle cell has an incomplete basal lamina covering (bottom left) (bar = 1 Pm).
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FIGURE 6. Rabbit soleus after 4-week suspension. (a) Semithin cross section. Surviving muscle fibers are small in size and display a jagged profile, while numerous blobs of fat infiltrate the endo- and perimysial space (bar = 40 pm). (b) Semithin longitudinal section. A lean muscle fiber with a villous profile shows almost regular sarcomeric pattern, while other fibers appear to be reduced to linear collections of myonuclei (bar = 40 pm).
Reduced Length Damages Soleus
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FIGURE 7. Rabbit soleus after 2-week suspension with the feet dorsiflexed. Semithin sections. (a) A muscle fascicle with polygonal fibers and normal endomysial space. (b) Myofibrils have a normal sarcomeric pattern (bar = 10 pm).
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ment associated with loss of Z-bands and mitotral core formations in the tenotomized rat soleus chondria.2922 seems to be part of the reparative process reconIn stage 11, muscle damage is different in that stituting the continuity of the sarcomeric banding it affects all components of the myofibers and it is pattern until normal fiber histology is restored.3 widespread rather than focal. Sarcomeres are vanFinally, rare fibers adapt to HS in rabbit soleus, ishing and the whole myofiber loses myofibrillar showing normal myofilament overlap and proper and sarcoplasmic fractions. Atrophy proceeds sarcomeric length, while the soleus remains at a faster in the fiber inner compartment than in the substantially shortened length. Why significant replasmalemma. parative changes are not expressed or are inhibIf HS persists for 4 weeks, atrophy of the soited in the unloaded rabbit soleus is unexplained. leus progresses to an extreme state in which part A species-specific characteristic can be advocated of the fibers are reduced to nuclear chains proin considering the sensitivity of rabbit soleus to vided with undifferentiated sarcoplasm. 'These unloaded shortening. cells share features with the so-called s a r c ~ c y t e s . ~ ~ Although muscle regeneration clearly develops This complex process does not seem to be susafter the second week of HS, the regenerative tained by lysosomal proteolysis in rabbit, because process does not counterbalance the progressive autophagic vacuoles were not observed in atrophic destruction of myofibrils, because regenerated fifibers. Fast atrophy of the soleus during HS has bers are still small and few at stage 111. The lack been suggested to be secondary to increased of' passive stretch during HS affects probably also ubiquitin-dependent protein degradation in rat." the growth of' regenerating fibers. On the other hand, the wasted muscle tissue is reChronic blood congestion obviously occurs in placed mainly by proliferated adipocytes, accountthe venous system of suspended hindlimbs.20 The ing for the increase in mass of the suspended sopossibility that compromised blood flow, demonstrated in suspended hindlimbs,14 affects the releus between 2- and 4-week suspensions. parative process of damaged soleus fibers, cannot The microscopic changes show evidence that be ruled out with certainty. the loss of sarcomeres in the soleus progresses to The morphological changes observed in axon involve the entire observable course of the fibers, terminals of suspended rabbit soleus appear to be probably beyond the limit required for the fibers mild and evident when soleus atrophy is already to adjust to the shorter length determined by HS.2 marked. They are probably retrograde in type or In the atrophic soleus fibers of suspended rabbit secondary to the severe atrophy of the dependent there are no significant signs of a reactive commuscle fibers. In the unloaded rat soleus, neuropensatory process. The nuclei appear quiescent, muscular junctions have been reported to be norlocalized in the subsarcolemmal region in most of ma1.20 the fibers, while ribosomes and sarcoplasmic reticUnlike the soleus, the medial and lateral porulum are scanty. 'Therefore, indirect signs of intion of gastrocnemius show only moderate atrocreased protein synthesis in atrophic fibers are phy affecting both type I and type I1 fibers with lacking. no alteration of the histoenzymatic patterns, even The similarities between muscle changes deterafter 4-week HS (unpublished observations). mined by HS or by tenotomy in rabbit soleus are noteworthy because tenotomized muscle displays Effects of Dorsiflexion. Avoiding hindlimb posmyofibrillar derangement, severe atrophy, and fat tural changes in the suspended rabbit prevents sosubstitution after several The two exleus muscle damage. It is surprising that dorsiflexperimental conditions determine a comparable bioion of the feet and sporadic contractions against mechanical change for the soleus consisting o f elastic resistance effectively prevent, not only myoseverely reduced working length and phasic confibrillar breakdown, but also muscle atrophy in traction without a load.'" unloaded hindlimbs. Stretching the hypoactive soContrary to that observed in rabbit, a better leus fibers is probably suf-ficient, at least for a few adaptation to sustained shortening, secondary to weeks, in preserving the balance between protein immobilization or tenotomy, takes place in rat and synthesis and degradation, despite the lack of cat soleus."" It first comprises a decrease in the load-bearing activities. number of sarcomeres in the midbelly region and, The type of foot dorsiflexion we used seems subsequently, a fast reorganization of myofibrillar pertinent to the specific biomechanical conditions apparatus, eventually attaining optimal myofiladetermined both by HS and hypogravity, in which ment o~erlap."'~The typical appearance of cen-
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there is a lack of resistance to the contraction of antigravity muscles.22 By applying an elastic resistance to shortening of antigravity muscle during HS, hindlimbs were neither immobilized nor engaged in supporting body wei ht as is the case in other experimental models. 10f1313 In our experimental approach, namely in group B rabbits, the specific working length of the soleus is, to some extent, respected during HS: in the resting posture, the soleus is fully stretched, while it becomes shortened against resistance during the plantar extension of the hindfeet. Such a simple device can be easily adapted to the feet of astronauts. It would have a preventive effect also on hypogravityinduced soleus muscle atrophy. 12s19-21 T h e new insight resulting from the above discussion is that an hypothesis can be formulated regarding the sensitivity of the soleus to HS. It is a tonic antigravity muscle, composed chiefly or almost exclusively of type I fibers, provided with distinctive biochemical and ultrastructural features such as elevated calcium exchange and characteristically wide Z-lines.",' Physiologically, during quadrupedal standing, the soleus muscle is stretched and, at the same time, it appears to be close to maximal and virtually continuous activity, whereas it is inactive and electrically silent during the nonstance phase of a ~ t e p . ' , ~Thus, ~ ' ~ ,tonic ~~ muscle contractions of the soleus are, in general, isometric rather than isotonic. This implies that soleus muscle myofibrils usually develop maximum contractile activities with little or no sliding of myofilaments. Conversely, by removing the constant tension imposed by load-bearing activity, isotonic contraction may occur in the soleus, but without important transfer of energy. Electromyographic recordings in rats support the hypothesis that an active shortening may take place in suspended soleus. Thus, soleus myofibrils can develop tonic and phasic contractions in an abnormally shortened state, compromising the intermyofilament bridges and the architecture of myofilament overlap and of the myofiber cytoskeleton. The shorter is the soleus and the longer its permanence in such a state, the greater the myofibrillar damage. In conclusion, isometric muscle contraction represents the optimal working condition for the soleus and, at the same time, may guarantee maintenance of its integrity. When this condition is subverted, tension-related myofibrillar damage can be expected. This may apply to a variety of pathologic conditions involving the activation of the fibers either in an abnormally shortened or
'.*'
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lengthened state, such as hypogravity, tenotomy, immobilization, spasticity due to CNS disorders, and lengthening contractions .3,722,1 871972 53
'
'
REFERENCES 1. Alford EK, Roy RR, Hodgson JA, Edgerton VR: Electro-
of rat soleus, medial gastrocnemius, and tibialis anterior during hind limb suspension. Exp Neurol 1987;96:635-649. 2. Anzil AP, Sancesario G, Massa R, Bernardi G: Myofibrillar disruption in the rabbit soleus muscle after one-week hindlimb suspension. Muscle Nerve 1991;14:358-369. 3. Baker JH: Segmental necrosis in tenotomized muscle fibers. Muscle Nerve 1983;6:29-39. 4. Bergman RA, Afifi AK: T h e structure of the rabbit soleus muscle and the structural alterations resulting from tenotomy.John Hopkinr MedJ 1969;124:119-131. 5 Corley K, Kovalchuk N , McComas AJ: Contrasting effects of suspension on hind limb muscles in the hamster. Exp Neurol 1984;85:30-40. 6 Everts ME, Clausen T : Effects of thyroid hormones on calcium contents and 45Ca exchange in rat skeletal muscle. Am J Physiol 1986;251:E258-E265. 7 Ferguson A, Vaugan L, Ward L: A study of disuse atrophy of skeletal muscle in the rabbit. J Bone Joint Surg 1957;39:583- 596. 8. Gardiner KF, Gardiner PF, Edgerton VR: Guinea pig soleus and gastrocnemius electromyograms at varying speeds, grades and loads. J Appl Physiol 1982;52:451-457. 9. Gauthier GF: Some ultrastructural and cytochemical features of fiber populations in the soleus muscle. Anat Rec 1975; 18O:551-564. 10. Graham SC, Roy RR, West SP, Thomason D, Baldwin KM: Exercise effects on the size and metabolic properties of soleus fibers in hindlimb-suspended rats. Auial Space Environ Med 1989;60:226-234. 11. Herbert ME, Roy RR, Edgerton VR: Influence of oneweek hindlimb suspension and intermittent high load exercise on rat muscles. Exp Neurol 1988;102:190- 198. 12. Ilyina-Kakueva EI, Portugalov VV, Krivenkova P: Space Hight effects on the skeletal muscles of rats. Aviat Space Enuiron Med 1976;47:700-703. 13. Jaspers SR, Fagan JM, Satarug S, Cook PH, Tischerl ME: Effects of immobilization on rat hind limb muscles under non-weight-bearing conditions. Muscle Nerve 1988; 11:458466. 14. LeBlanc A , Marsh C;, Evans H, Johnson P, Schneider V, Jhingran S: Bone and muscle atrophy with suspension of the rat. J Appl Physiol 1985;58:1669- 1675. 15 McMinn RM, Vrbova G: Morphological changes in red and pale muscles following tenotomy. Nature 1962; 195:509. 16 McMinn RM, Vrbova G: Motoneurone activity as a cause of degeneration in the soleus muscle of the rabbit. Q J Exp Physiol 1967;52:4 1 1- 4 15. 17 Musacchia XJ, Deavers DR, Meininger GA, Davis TP: A model for hypokinesia: Effects on muscle atrophy in the rat. J Appl Physiol 1980;48:479-486. 18 Ogilvie RV, Armstrong RB, Baird KE, Bottoms CL: Lesion in the rat soleus muscle following eccentrically biased exercise. Am J Anat 1988; 182:335-346. 19. Riley DA, Ellis S, Slocum GK, Satyanarayana T, Bain JLW, Sedlak FR: Hypogravity-induced atrophy of rat soleus and extensor digitorum longus muscles. Muscle Nerue 1987; 1 O:56O- 568. 20. Riley DA, Slocum GR, Bain JLW, Sedlak FR, Sowa TE, Mellender J W: Rat hindlimb unloading: soleus histochemImyography
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21.
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