Skeletal muscle adaptation flown on Cosmos 1667
in rats
D. DESPLANCHES, M. H. MAYET, E. I. ILYINA-KAKUEVA, B. SEMPORE, AND R. FLANDROIS Unit6 Associ& Centre National de lu Recherche Scientifique 1341, Laboratoire de Physiologie, Fucultk de M6decine Lyon Grange-Blanche, Universitk Claude Bernard Lyon I69373, Lyon Cedex 08, France; and Institute of Biomedical Problems, Moscow 123007, USSR DESPLANCHES, D., M. H. MAYET, E. I. ILYINA-KAKUEVA, B. SEMPORE, AND R. FLANDROB. Skeletal muscle adaptation in ruts flown on Cosmos1667. J. Appl. Physiol. 68(l): 48-52, 1990.-Seven male Wistar rats were subjected to 7 days of
primary muscular alterations in the earlier period of exposure to microgravity. Besides the three main fiber types classically described by Brooke and Kaiser (3), we examined the intermediate fibers corresponding to the weightlessnesson the Soviet biosatellite Cosmos1667.Muscle transition from one fiber type to another. Because the histomorphometry and biochemical analyses were performed alterations in enzyme activities reflect adaptive changes on the soleus(SOL) and extensor digitorum longus (EDL) of in the capacity of metabolic pathways, lactate dehydroflight rats (group Fj’ and comparedwith data from three groups genase (LDH), citrate synthase (CS), and 3-hydroxyacylof terrestrial controls: one subjectedto conditions similar to CoA dehydrogenase (HAD) were determined in addition group F in spaceexcept for the state of weightlessness(group S) and the others living free in a vivarium ( Vr, Vg). Relative to to muscle capillarization. The low number of spaceflights demonstrates the need group V2 (its ageand weight-matched control group), group F showeda greater decreaseof musclemassin SOL (23%) than to develop Earth-bound models simulating microgravity. in EDL (11%). In SOL a decreasein the percentageof type I To mimic weightlessness, several models such as hindfibers was counterbalancedby a simultaneousincreasein type limb immobil&ation (2) or hindlimb suspension (8, 17, IIa fibers. The cross-sectionalarea of type I fiber was reduced 18) have recently been developed. In this report the by 24%. No statistically significant difference in capillarization effects of weightlessness induced by either a 7-day Cosand enzymatic activities was observedbetween the groups. In EDL a reduction in type I fiber distribution and 3-hydroxyacyl- mos 1667 flight or 1 wk of tail suspension are directly CoA-dehydrogenaseactivity (27%) occurred after the flight. compared. The small histochemical and biochemical changes reported suggestthe interest in studying muscular adaptation during a flight of longer duration. spaceflight; histochemistry; capillarization; enzyme activity
EXPOSURETO MICROGRAVITY elicits atrophy of selected
muscles and loss of skeletal mass (4,13,14,24,30). Thus the Cosmos biosatellites afforded an excellent opportunity to study the effects of weightlessness on the locomotor system (12). The synchronous experiment on Earth that reproduces all the physiologically significant factors of spaceflight except weightlessness makes possible a comparative analysis of the state of the animals under conditions of spaceflight and on Earth. In the previous Cosmos biosatellite experiments, Ilyina-Kakueva et al. (13) and Kazarian et al. (14) reported a greater decrease in the weight of the rat soleus muscle (SOL), a slow-twitch muscle containing predominantly slowtwitch oxidative fibers (type I) than in the extensor digitorum longus (EDL) containing fast-twitch oxidative glycolytic (IIa) and fast-twitch glycolytic (IIb) fibers. Atrophy of selected muscles was described in long-term spaceflight (previous Cosmos missions lasted 18-22 days), but space missions are often shorter. Therefore the purpose of this work was to study the 48
MATERIALS AND METHODS
Animals. The experiments were performed on male Wistar rats (SPF colony) weighing 330-350 g. Seven rats flown on Cosmos 1667 were decapitated 4-8 h after a 7day spaceflight. Aboard the biosatellite, the rats were kept in individual cylindrical (9.5 cm diam, 28 cm long) cages. To determine confinement-induced effects, a synchronous group was exposed on Earth to conditions similar to those of the flight group in space, except for the state of weightlessness. The existence of a third and fourth group (vivarium 1 and 2) was justified by the fact that the synchronous experiment took place 1 mo later than the flight experiment. Vivarium 1 was the age- and weight-matched control for the synchronous group, and vivarium 2 was the control for the flight group. Groundbased controls (vivarium 1 and 2) were housed individually in the animal quarters at the Institute of Biomedical Problems (Moscow), where the temperature (24°C) and light-dark cycle (12:12 h) were regulated. They received a pastelike diet once a day, whereas the flight group received an identical diet four times per day at 6h intervals. Water was provided ad libitum. Four SOL and seven EDL muscles were dissected out for histochemical and biochemical analyses, frozen in liquid nitrogen, and stored at -7OOC. In each group, only four SOL muscles were available for study.
0161-7567/90 $1.50 Copyright 0 1990 the American Physiological Society
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WEIGHTLESSNESS
AND
HistochemicaZ analysis. The samples were mounted in an embedding medium (TEK ACT compound), frozen in isopentane cooled to its freezing point, and stored at -80°C until analysis was performed. Serial sections (10 pm thick) of frozen muscle blocks were cut on a microtome at -3OOC. Fiber types were stained for the myosin adenosinetriphosphatase (ATPase) method (9, 11) after preincubation at pH 4.4 for 4 min at 25°C in acid buffer (50 mM acetic acid-25 mM CaClJ. The ATPase reaction was then carried out in a buffer (18 mM Ca&-2.7 mM ATP) for 20 min (pH 9.4) at 37°C. Muscle fibers are classified into three major types (I, IIa, IIb) and intermediate fiber types (Int I and II) (5, 6, 21). The histochemical method used was based on observed differences in pH lability of the myosin ATPase activity of the isomyosins in the different fibers and validated by a comparative immunohistochemical and enzyme histochemical analysis (9, 21). Fiber-type distribution is expressed as the number of fibers of each type relative to the total number of fibers (200 fibers/section). Capillaries were visualized by means of the ATPase technique after preincubation at a pH of 4.0 (28). Capillaries per fiber and capillary density were determined as described by Andersen and Henriksson (1). Enzyme activities. The tissues were homogenized in a 0.3 M phosphate buffer containing 0.05% bovine serum albumin at pH 7.7. To disrupt the mitochondrial membrane, homogenates were frozen and thawed three times. CS (EC 4.1.3.7) was determined following the method of Srere (25). LDH (EC 1.1.1.27) and HAD (EC 1.1.1.35) measurements were based on NAD-NADP-dependent enzymatic fluorimetric assays (15). Enzyme activities are expressed as micromoles substrate per minute per gram wet weight. Statistical analysis. The intergroup differences were assessed by means of the Mann-Whitney sum-rank test. All data are expressed as means t SE. In all cases the level of significance was set at P < 0.05. RESULTS Muscle atrophy. The decrease in total muscle mass during the Cosmos 1667 flight was 23 and 11% for the SOL and EDL muscles, respectively, compared with vivarium 2 (Table 1). The data in Table 1 show that, despite the additional month of age, the muscle mass in vivarium 1 and the synchronous group was not significantly greater than in vivarium 2. Histochemistry. In the SOL, after 7 days of weightlessness, a decrease in the percentage of type I fibers (from 89 to 79%), which appear dark, was counterbalanced by TABLE
1. Muscle wet weights Group Vivarium
SOL EDL
169.Ok4.5 170.3&3.1
1
Synchronous
Vivarium
172.4k6.0 174.6t4.6
158.4k4.5 165.7k7.8
2
Flight 122.4&O* 146.7&&O*
Values are means + SE for soleus (SOL; n = 4 for each group) and extensor digitorum longus (EDL; n = 7 for each group). * Significantly different from vivarium 1 and 2 and synchronous groups.
MUSCLE
49
ATROPHY
a simultaneous significant increase in type IIa fibers (from 4 to 14%), which appear light on the section; intermediate type I fibers appeared as gray with no change in their number (7%) (Fig. 1). Neither capillary density (-900 capillaries/mm2) nor capillaries per fiber (2.7 capillaries/fiber) showed a significant difference across the four groups (see Table 3). In the EDL, the percentage of type I fibers significantly decreased from 10 to 5% a&er the Cosmos flight (Table 2). A similar finding was noted between the synchronous and flight groups. Enzyme actiuities. EDL showed higher activities of LDH (505 t 39 pmol min-’ . g-‘) than SOL (Table 3). CS activity, a marker of Krebs turnover, remained constant (35 pmol min-l . g-‘) whatever the muscle. HAD activity, which reflects free fatty acid oxidation, was higher in SOL (13.2 & 1.6 prnol. min-’ *g-l) than in EDL (6.9 t 0.8 prnol min-’ g-l). No difference was found among the four groups, except for HAD activity, which was decreased after flight in the EDL (Table 3). l
l
l
DISCUSSION
Muscle atrophy induced by microgravity is correlated with the degree of the load-bearing function (12). Skeletal muscle alteration occurs markedly in slow-twitch compared with fast-twitch muscles. After Cosmos biosatellite 605 and 690 flights, Chui and Castleman (4) reported a decrease in SOL mass (22 and 25%, respectively). In rats flown on Cosmos 782 and 1129, the exposure to weightlessness led to a greater decline in SOL weight as it reached 38 and 55%, respectively. During these flights, rats were exposed to 18.5-21.5 days of microgravity. The effect of a short-term spaceflight (current space missions) was until now poorly understood. The SOL muscle weight of rats placed in orbit for 7 days aboard Cosmos 1667, compared with vivarium 2 was decreased by 23%, whereas atrophy of the EDL muscle reached 11%. Muscle weight decrements during Cosmos 1667 were similar to those reported during the Spacelab- shuttle flight where the mass of the SOL and EDL muscles was decreased by 36 and 16%, respectively (10, 16). They confirmed the greater atrophy occurring in the SOL muscle after 1 wk of hindlimb tail suspension (37%) (5,6) or harness suspension (29%) (18,19). However, the loss of EDL mass was greater than after 1 wk of Morey’s tail suspension model, since it reached only 4% in the latter case (5). In SOL microgravity induced a slight tendency to a decreased type I fiber percent distribution (11%) counterbalanced by a large significant increase in type IIa fibers. We had only four SOL muscles at our disposal to perform the study, and this may account for the nonsignificant decrease of type I fiber percentage. The effects of the present flight seem to have occurred earlier than those of the tail suspension technique. Indeed, type IIa fiber distribution was significantly increased only after 2 wk of hindlimb suspension (5). After 1 wk a decrease in the percent distribution of type I fibers (85.5 t 2.4 vs. 73.8 $- 2.1%) had b een evidenced, whereas intermediate
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WEIGHTLESSNESS
50
AND MUSCLE
ATROPHY
FIG. 1. Cross sections of rat soleus muscle stained for myofibrillar ATPase activity after preincubation at pH 4.4. A: vivarium 2 group. I, type I muscle fiber; Int I, intermediate type I muscle fiber; IIa, type IIa muscle fiber. B: decrease in percentage and cross-sectional area of type I fibers with concomitant significant increase in percentage of type IIa fibers in flight groups.
TABLE
2. Influence of weightlessness on percent distribution
of fiber type in SOL and EDL % Distribution
SOL (n = 4) I
Int I
EDL (n = 7) IIa
I
II
Vivarium 1 85.5k3.4 4.4k1.6 lO.lk3.9 9.5kO.7 Synchronous 84.5k3.4 8.Ok3.1 7.5f1.9 9.3*0.4* Vivarium 2 88.5k1.7 7.5k1.2 4.OkO.6 lO.Ozb1.7 Flight 78.5k3.4 7.9k3.1 13.6f3.7t 5.3+0.7t Values are means + SE. SOL, soleus; EDL, extensor digitorum longus. Significantly t between vivarium 2 and flight groups. TABLE
3. LDH,
CS, and HAD activities and capillarization
22.321.6 15.9f2.9 19.8k3.3 16.1k2.1 different: *between
Int II
In,
3.6f1.7 64.6kO.8 5.1kl.O 68.Ok3.1 5.2f1.6 65.0k4.6 4.6k1.5 71.9k2.1 synchronous and flight groups,
in SOL and EDL Group
Vivarium 1 LDH, pmol . min-’ . g-’ CS, pm01 . min-’ +g-’ HAD, pmol . min-’ . g-i CD, capillaries/mm* Capillaries/fiber
124.Ok7.0 37.2k3.1 13.2k1.6 842.Ok156.0 2.7eO.4
Synchronous SOL (n = 4) 131.0f19.0 34.5k1.9 ll.Ok2.0 928.0f114.0 2.9f0.3
Vivarium 2
Flight
140.2f17.0 34.6k4.0 12.0+1.5 994.O-c81.0 2.9fO.l
llO.Ozk8.0 35.222.0 12.1kl.O 1,161.0f220.0 2.5f0.3
EDL (n = 7) LDH, rmol . min-’ .g-’ 505.Ok39.0 53o.ozk44.0 546.Ok28.0 528.Ok45.0 CS, pm01 . min-’ . g-’ 34.2zk2.9 30.6f2.5 33.Ok1.3 29.5f2.1 HAD, Fmol .min-’ . g-’ 6.9f0.8 6.5+0.4 7.0f0.8 5.1+0.5* Values are means + SE, n, no. of measurements. LDH, lactate dehydrogenase; CS, citrate synthase; HAD, 3-hydroxyacyl-CoA dehydrogenase; CD, capillary density; SOL, soleus; EDL, extensor digitorum longus. * Significantly different between vivarium 2 and flight groups.
type I fiber distribution was doubled (8.5 -C 1.9 vs. 17.1 -e 1.7%). Type IIa fiber distribution remained unchanged (5.9 + 1.2 vs. 7.3 -I 1.5%). In EDL, during the COSMOS 1667 flight, microgravity induced a reduction of type I fiber from 10 to 5%. Takacs et al. (27) had also observed an increase in the quantity of myosin light chain-3 subunits in the SOL and EDL muscles of rats exposed to weightlessness for 18.5 days (Cosmos 1129). Martin et al. (16) reported an increase in the percentage of dark
ATPase fibers in Spacelab- flight muscles with a predominance of light ATPase fibers. According to our previous experiments (5), the slow-twitch type I fiber becomes a hybrid fiber like the intermediate fiber, representing transitional stages within the steps of the major fiber types (5). The reports of Fitts et al. (8) and Reiser et al. (23) also suggested that the increase in the mean maximal velocity of SOL muscle shortening after hindlimb suspension was associated with an increased expres-
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WEIGHTLESSNESS
AND MUSCLE
sion of a second faster migrating isozyme of myosin, the amount of fast-type myosin heavy chain increasing with relatively small changes in the myosin light chain composition. The findings of Templeton et al. (29) are consistent with our earlier results, indicating that chronic reduction of gravitational load preserves total fiber number and induces a shift in fiber types rather than simultaneous type I fiber loss and type II fiber synthesis, according to the absence or presence of embryonic or neonatal myosin. Steffen and Musacchia (26) also suggested that unaltered DNA contents after a short-term exposure to weightlessness indicated no loss of muscle fibers. After 7 days of microgravity (Cosmos 1667), neither mean capillary density nor capillaries per fiber were affected in SOL or EDL. These data are at variance with those of Musacchia et al. (20), who reported a significant increased density of capillaries (50%) in SOL muscles submitted to 7 days of weightlessness (Spacelab-3). In an earlier experiment, we showed a decrease of 46% of capillaries per fiber after hindlimb suspension but it was after a longer period (5 wk). These modifications of capillary density (20) or capillaries per fiber (5) raise the question of a potential alteration in blood flow to atrophied muscles as suggested by Musacchia et al. (20). No alteration in functional capacity of rat muscles was noted after the Cosmos 1667 flight except in EDL, where a decrease in HAD activity (27%) was observed. In SOL, the absence of reductions in both CS and HAD activities, after 1 wk of weightlessness, is in agreement with our previous observations (5). Indeed, 5 wk of hindlimb suspension were necessary to induce a decrease in these activities (23 and 39%, respectively). This is in contrast with the findings of Fell et al. (7), who observed a reduction in CS activity after 1 wk of harness suspension. In SOL and EDL, the nonaltered LDH activity was identical to that observed after 1 wk of hypokinesiahypodynamia (5). However, after a longer flight (21.5 days, Cosmos 605), Portugalov and Petrova (22) reported alterations in the spectrum of LDH isozymes in favor of an increased glycolytic process. With respect to free fatty acid oxidation, the decrease in HAD activity observed in EDL was discordant with results reported after 1 wk of suspension (5) but was confirmed after 12.5 days for the Cosmos 1887 flight (unpublished data). A maintenance of oxidative enzymatic activity (CS and HAD) had been observed after 7 days for Cosmos 1667. These data are similar to those demonstrated in SOL and EDL muscles by Martin et al. (16) from the Spacelab 3 mission. An interesting point was advanced in their study. To explain the absence of variation in succinic dehydrogenase (SDH) activity, they suggested that the total amount of the oxidative enzyme per fiber (product of cross-sectional area and SDH activity) should be determined. Thus they took into account that a preferential loss of muscle volume (and probably contractile protein) relative to metabolic enzymes may have occurred. Martin et al. (16) argued that their results (increased SDH activity while the total amount of the SDH enzyme per fiber decreased by 21%) point to an interaction between the rate of synthesis and degradation of specific proteins and the
ATROPHY
51
control of cell volume after microgravity. What determines the differential responses of slowand fast-twitch fibers to weightlessness remains an unresolved problem. Martin et al. (16) suggested that the decrease in mechanical loading of muscle may have a more important role than the total amount of electromyographic activity per day because the SOL during suspension regains its normal pattern of activity within 7 days. Furthermore, the role of hormones in the development of muscular atrophy and the slow-to-fast alterations in skeletal muscle fibers should not be underestimated. For example, the growth of fast-twitch fibers is regulated by insulin (3 1), triiodothyronine stimulates the growth of both slow- and fast-twitch muscles, and @adrenergic agonists like clenbuterol (31) cause hypertrophy of fast-twitch fibers in SOL and EDL muscles accompanied by slow-to-fast fiber conversion. A higher concentration of corticosterone and a lower concentration of thyroxine in blood was reported after flight, showing the emergence of an acute gravitational stress after flight. In conclusion, histochemical changes show that muscular atrophy is accompanied by an increased expression of fast-type myosin in the slow-type I fiber in SOL. No variation in capillarization or in the three enzyme activities was observed except a decrease in HAD for the EDL. Similar tendencies in groups S and F indicate that muscular alterations reflect the effect of weightlessness rather than confinement. The present observations are consistent with those obtained in the tail suspension model, suggesting the interest of ground-based simulations to study the effects of microgravity on muscle. The small structural and functional muscular adaptations reported after short-term exposure to weightlessness demonstrate the need to study muscular adaptations during a flight of longer duration. The authors are grateful to the staff of the Institute of Biomedical Problems, especially 0. G. Gazenko and Dr. E. A. Ilyin. They thank Dr. V. S. Oganov and V. S. Skuratova for providing the muscle samples used in these studies. They also thank Dr. M. J. Carew for reviewing the English manuscript. This work was supported by a grant from Centre National d’Etudes Spatiales. Address for reprint requests: D. Desplanches, UA CNRS 1341, Laboratorie de Physiologie, Lyon Grange-Blanche Universitit, 69373 Lyon Cedex 08, France. Received 21 February 1989; accepted in final form 30 August 1989. REFERENCES Capillary supply of the quad1. ANDERSEN, P., AND J. HENRIKSSON. riceps femoris muscle of man: adaptive response to exercise. J. Physiol. Lord 270: 667-690, 1977. 2. BOOTH, F. W., AND J. R. KELSO. Effect of hindlimb immobilization on contractile and histochemical properties of skeletal muscle. Pfhegers Arch. 342: 231-238, 1973. 3. BROOKE, M. H., AND K. K. KAISER. Muscle fiber types: how many and what kind? Arch. Neural. 23: 369-379, 1970. 4. CHUI, L. A., AND K. R. CASTLEMAN. Morphometric analysis of rat muscle fibers following spaceflight and hypogravity. Pkysiologist 23, Suppl.: S576-S578,1980. 5. DESPLANCHES, D., M. H. MAYET, B. SEMPORE, AND R. FLANDROIS. Structural and functional responses to prolonged hindlimb suspension in rat muscle. J. Appl. Physiol. 63: 558-563, 1987. 6. DESPLANCHES, D., M. H. MAYET, B. SEMPORE, J. FRUTOSO, AND R. FLANDROIS. Effect of spontaneous recovery or retraining after
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52 hindlimb suspension 1743,1987. 7. FELL, R. D., J. M.
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STEFFEN, AND X. J. MUSACCHIA. Effect of hypokinesia-hypodynamia on rat muscle oxidative capacity and glucose uptake. Am, J. Physiol. 249 (Regulatory Integrative Comp. Physiol. 18): R308-R312, 1985. 8. FITTS, R. H., J. M. METZGER, D. A. RILEY, AND B. R. UNSWORTH. Models of disuse: a comparison of hindlimb suspension and immobilization. J. Appl. Physiol. 60: 1946-1953, 9. GRANDMONTAGNE, M., 0. VAAGE, N. KOPKE HERMANSEN. Confrontation de mbthodes
1986. VOLLESTAD, AND L. histochimiques et
d’immunofluorescence pour le typage des fibres du muscle squelettique de rat (Abstract). J. Physiol. Paris 78: l4A, 1982. 10. GRINDELAND, R., T. FAST, M. RUDER, M. VASQUES, P. LUNDGREN, S. SCIBETTA, J. TREMOR, P. BUCKENDAHL, L. KEIL, 0. CHEE, T. REILLY, B. DALTON, AND P. CALLAHAN. Rodent body, organ and muscle weight response to seven days of microgravity (Abstract). Physiologist 28: 375, 1985. 11. HERMANSEN, L., N. K. VOLLESTAD, P. H. STAFF, 0. A. DALJORD, AND 0. GRONNEROND. The effect of immobilization and training on strength and composition of human skeletal muscle. In: Space PhysioZogy/PhysioZogie SputiaZe. Lyon, France: CNRS, 1983, p. 255-266. 12. ILYIN, E. A. Investigations on biosatellites of the Cosmos series. Aviat. Space Environ. Med. 54, Suppl. 1: S9-S15,1983. 13. ILYINA-KAKUEVA, E. I., V. V. PORTUGALOV, AND N. P. KRIVENKOVA. Space flight effects on the skeletal muscles of rats. Aviat. Space Environ. Med. 47: 700-703, 1976. 14. KAZARIAN, V. A., E. A. RAPOPORT, L. A. GONCHAROVA, AND S. I. BALYCHEVA. Effects of prolonged weightlessness on protein metabolism in rat and white skeletal muscles. Kosm. Biol. Aviakosm. Med. 11: 19-23,1977. 15. LOWRY, 0. H., AND J. V. PASSONNEAU. A Flexible System of Enzymatic Analysis. New York: Academic, 1973. 16, MARTIN, T. P., V. R. EDGERTON, AND R. E. GRINDELAND. Influence of spaceflight on rat skeletal muscle. J. AppZ. Physiol. 65: 2318-2325,1988. 17. MOREY, E. R. Spaceflight and bone turnover: correlation with a new rat model of weightlessness. Bioscience 29: 168-172,1979. 18. MUSACCHIA, X. J., D. R. DEAVERS, G. A. MEININGER, AND T. P. DAVIS. A model for hypokinesia: effects on muscle atrophy in the rat. J. Appl. Physiol. 48: 479-486, 1980. 19. MUSACCHIA, X. J., J. M. STEFFEN, AND D. R. DEAVERS. Rat hindlimb muscle responses to suspension hypokinesia/hypodyna-
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mia. Aviat. Space Environ. Med. 54: 1015-1020, 1983. 20. MUSACC!-IIA, X. J., J. M. STEFFEN, R. D. FELL, AND M. J. DOMBROWSKI. Comparative morphometry of fibers and capillaries in soleus following weightlessness (SL-3) and suspension. Physiologist 31: S28-S29,1988. 21. PIEROBON-BORMIOLI, S., S. SARTORE, L. DALLA LIBERA, M. VITADELLO, AND S. SCHIAFFINO. “Fast” isomyosins and fiber types in mammalian skeletal muscle. J. Histochem. Cytochem. 29: 11791188,198l. 22. PORTUGALOV, V. V., AND N. V. PETROVA. LDH isoenzymes of skeletal muscles of rats after spaceflight and hypokinesia. Aviut. Space Environ. Med. 47: 834-838, 1976. 23. REISER, P. J., C. E. KASPER, AND R. L. Moss. Myosin subunits and contractile properties of single fibers from hypokinetic rat muscles. J. Appl. Physiol. 63: 2293-2300, 1987. 24. RILEY, D. A., S. ELLIS, G. R. SLOCUM, T. SATYANARAYANA, J. L. W. BAIN, AND F. R. SEDLAK. Morphological and biochemical changes in soleus and extensor digitorum longus muscles of rats orbited in Spacelab 3. Physiologist 28: S207-s208, 1985. 25. SRERE, P. A. Citrate synthase. Methods Enzymol. 13: 3-5,1969. 26. STEFFEN, J. M., AND X. J, MUSACCHIA. Spaceflight effects on adult rat muscle protein, nucleic acids, and amino acids. Am. J. Physiol. 251 (Regulatory Integrative Comp. Physiol. 20): R1059-R1063, 1986. 27, TAKACS, O., MI RAPCSAK, A. SZOOR, V. S. OGANOV, T. SZILAGYI, S. S. OGANESYAN, AND F. GUBA. Effect of weightlessness on myofibrillar proteins of rat skeletal muscles with different functions in experiment of Biosatellite “Cosmos 1129.” Actu Physiol. Hung. 62: 228-233,1983. 28. TAMAKI, N. Effect of growth on muscle capillarity and fiber type composition in rat diaphragm. Eur. J. AppZ. Physioh &cup. Physiol. 54: 24-29, 1985. 29. TEMPLETON, G, H., H. L, SWEENEY, B. F. TIMSON, M. PADALINO, AND G. A. DUDENHOEFFER. Changes in fiber composition of soleus muscle during rat hindlimb suspension. J. AppZ. Physiol. 65: 11911195,1988. 30. WRONSKI, T. J., E. R. MOREY-HOLTON, S. B. DOTY, A. C, MAESE, AND C. C. WALSH. Histomorphometric analysis of rat skeleton following spaceflight. Am. J. Physiol. 252 (Regulatory Integrative Comp. Physiol. 21): R252-R255, 1987. 31. ZEMAN, R. J., R. LUDEMAN, T. G. EASTON, AND J. D, ETLINGER.
Slow to fast alterations in skeletal muscle fibers caused by clenbuterol, a Bz-receptor agonist. Am. J. Physiol. 254 (Endocrinol. Metab.
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in rats
D. DESPLANCHES, M. H. MAYET, E. I. ILYINA-KAKUEVA, B. SEMPORE, AND R. FLANDROIS Unit6 Associ& Centre National de lu Recherche Scientifique 1341, Laboratoire de Physiologie, Fucultk de M6decine Lyon Grange-Blanche, Universitk Claude Bernard Lyon I69373, Lyon Cedex 08, France; and Institute of Biomedical Problems, Moscow 123007, USSR DESPLANCHES, D., M. H. MAYET, E. I. ILYINA-KAKUEVA, B. SEMPORE, AND R. FLANDROB. Skeletal muscle adaptation in ruts flown on Cosmos1667. J. Appl. Physiol. 68(l): 48-52, 1990.-Seven male Wistar rats were subjected to 7 days of
primary muscular alterations in the earlier period of exposure to microgravity. Besides the three main fiber types classically described by Brooke and Kaiser (3), we examined the intermediate fibers corresponding to the weightlessnesson the Soviet biosatellite Cosmos1667.Muscle transition from one fiber type to another. Because the histomorphometry and biochemical analyses were performed alterations in enzyme activities reflect adaptive changes on the soleus(SOL) and extensor digitorum longus (EDL) of in the capacity of metabolic pathways, lactate dehydroflight rats (group Fj’ and comparedwith data from three groups genase (LDH), citrate synthase (CS), and 3-hydroxyacylof terrestrial controls: one subjectedto conditions similar to CoA dehydrogenase (HAD) were determined in addition group F in spaceexcept for the state of weightlessness(group S) and the others living free in a vivarium ( Vr, Vg). Relative to to muscle capillarization. The low number of spaceflights demonstrates the need group V2 (its ageand weight-matched control group), group F showeda greater decreaseof musclemassin SOL (23%) than to develop Earth-bound models simulating microgravity. in EDL (11%). In SOL a decreasein the percentageof type I To mimic weightlessness, several models such as hindfibers was counterbalancedby a simultaneousincreasein type limb immobil&ation (2) or hindlimb suspension (8, 17, IIa fibers. The cross-sectionalarea of type I fiber was reduced 18) have recently been developed. In this report the by 24%. No statistically significant difference in capillarization effects of weightlessness induced by either a 7-day Cosand enzymatic activities was observedbetween the groups. In EDL a reduction in type I fiber distribution and 3-hydroxyacyl- mos 1667 flight or 1 wk of tail suspension are directly CoA-dehydrogenaseactivity (27%) occurred after the flight. compared. The small histochemical and biochemical changes reported suggestthe interest in studying muscular adaptation during a flight of longer duration. spaceflight; histochemistry; capillarization; enzyme activity
EXPOSURETO MICROGRAVITY elicits atrophy of selected
muscles and loss of skeletal mass (4,13,14,24,30). Thus the Cosmos biosatellites afforded an excellent opportunity to study the effects of weightlessness on the locomotor system (12). The synchronous experiment on Earth that reproduces all the physiologically significant factors of spaceflight except weightlessness makes possible a comparative analysis of the state of the animals under conditions of spaceflight and on Earth. In the previous Cosmos biosatellite experiments, Ilyina-Kakueva et al. (13) and Kazarian et al. (14) reported a greater decrease in the weight of the rat soleus muscle (SOL), a slow-twitch muscle containing predominantly slowtwitch oxidative fibers (type I) than in the extensor digitorum longus (EDL) containing fast-twitch oxidative glycolytic (IIa) and fast-twitch glycolytic (IIb) fibers. Atrophy of selected muscles was described in long-term spaceflight (previous Cosmos missions lasted 18-22 days), but space missions are often shorter. Therefore the purpose of this work was to study the 48
MATERIALS AND METHODS
Animals. The experiments were performed on male Wistar rats (SPF colony) weighing 330-350 g. Seven rats flown on Cosmos 1667 were decapitated 4-8 h after a 7day spaceflight. Aboard the biosatellite, the rats were kept in individual cylindrical (9.5 cm diam, 28 cm long) cages. To determine confinement-induced effects, a synchronous group was exposed on Earth to conditions similar to those of the flight group in space, except for the state of weightlessness. The existence of a third and fourth group (vivarium 1 and 2) was justified by the fact that the synchronous experiment took place 1 mo later than the flight experiment. Vivarium 1 was the age- and weight-matched control for the synchronous group, and vivarium 2 was the control for the flight group. Groundbased controls (vivarium 1 and 2) were housed individually in the animal quarters at the Institute of Biomedical Problems (Moscow), where the temperature (24°C) and light-dark cycle (12:12 h) were regulated. They received a pastelike diet once a day, whereas the flight group received an identical diet four times per day at 6h intervals. Water was provided ad libitum. Four SOL and seven EDL muscles were dissected out for histochemical and biochemical analyses, frozen in liquid nitrogen, and stored at -7OOC. In each group, only four SOL muscles were available for study.
0161-7567/90 $1.50 Copyright 0 1990 the American Physiological Society
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AND
HistochemicaZ analysis. The samples were mounted in an embedding medium (TEK ACT compound), frozen in isopentane cooled to its freezing point, and stored at -80°C until analysis was performed. Serial sections (10 pm thick) of frozen muscle blocks were cut on a microtome at -3OOC. Fiber types were stained for the myosin adenosinetriphosphatase (ATPase) method (9, 11) after preincubation at pH 4.4 for 4 min at 25°C in acid buffer (50 mM acetic acid-25 mM CaClJ. The ATPase reaction was then carried out in a buffer (18 mM Ca&-2.7 mM ATP) for 20 min (pH 9.4) at 37°C. Muscle fibers are classified into three major types (I, IIa, IIb) and intermediate fiber types (Int I and II) (5, 6, 21). The histochemical method used was based on observed differences in pH lability of the myosin ATPase activity of the isomyosins in the different fibers and validated by a comparative immunohistochemical and enzyme histochemical analysis (9, 21). Fiber-type distribution is expressed as the number of fibers of each type relative to the total number of fibers (200 fibers/section). Capillaries were visualized by means of the ATPase technique after preincubation at a pH of 4.0 (28). Capillaries per fiber and capillary density were determined as described by Andersen and Henriksson (1). Enzyme activities. The tissues were homogenized in a 0.3 M phosphate buffer containing 0.05% bovine serum albumin at pH 7.7. To disrupt the mitochondrial membrane, homogenates were frozen and thawed three times. CS (EC 4.1.3.7) was determined following the method of Srere (25). LDH (EC 1.1.1.27) and HAD (EC 1.1.1.35) measurements were based on NAD-NADP-dependent enzymatic fluorimetric assays (15). Enzyme activities are expressed as micromoles substrate per minute per gram wet weight. Statistical analysis. The intergroup differences were assessed by means of the Mann-Whitney sum-rank test. All data are expressed as means t SE. In all cases the level of significance was set at P < 0.05. RESULTS Muscle atrophy. The decrease in total muscle mass during the Cosmos 1667 flight was 23 and 11% for the SOL and EDL muscles, respectively, compared with vivarium 2 (Table 1). The data in Table 1 show that, despite the additional month of age, the muscle mass in vivarium 1 and the synchronous group was not significantly greater than in vivarium 2. Histochemistry. In the SOL, after 7 days of weightlessness, a decrease in the percentage of type I fibers (from 89 to 79%), which appear dark, was counterbalanced by TABLE
1. Muscle wet weights Group Vivarium
SOL EDL
169.Ok4.5 170.3&3.1
1
Synchronous
Vivarium
172.4k6.0 174.6t4.6
158.4k4.5 165.7k7.8
2
Flight 122.4&O* 146.7&&O*
Values are means + SE for soleus (SOL; n = 4 for each group) and extensor digitorum longus (EDL; n = 7 for each group). * Significantly different from vivarium 1 and 2 and synchronous groups.
MUSCLE
49
ATROPHY
a simultaneous significant increase in type IIa fibers (from 4 to 14%), which appear light on the section; intermediate type I fibers appeared as gray with no change in their number (7%) (Fig. 1). Neither capillary density (-900 capillaries/mm2) nor capillaries per fiber (2.7 capillaries/fiber) showed a significant difference across the four groups (see Table 3). In the EDL, the percentage of type I fibers significantly decreased from 10 to 5% a&er the Cosmos flight (Table 2). A similar finding was noted between the synchronous and flight groups. Enzyme actiuities. EDL showed higher activities of LDH (505 t 39 pmol min-’ . g-‘) than SOL (Table 3). CS activity, a marker of Krebs turnover, remained constant (35 pmol min-l . g-‘) whatever the muscle. HAD activity, which reflects free fatty acid oxidation, was higher in SOL (13.2 & 1.6 prnol. min-’ *g-l) than in EDL (6.9 t 0.8 prnol min-’ g-l). No difference was found among the four groups, except for HAD activity, which was decreased after flight in the EDL (Table 3). l
l
l
DISCUSSION
Muscle atrophy induced by microgravity is correlated with the degree of the load-bearing function (12). Skeletal muscle alteration occurs markedly in slow-twitch compared with fast-twitch muscles. After Cosmos biosatellite 605 and 690 flights, Chui and Castleman (4) reported a decrease in SOL mass (22 and 25%, respectively). In rats flown on Cosmos 782 and 1129, the exposure to weightlessness led to a greater decline in SOL weight as it reached 38 and 55%, respectively. During these flights, rats were exposed to 18.5-21.5 days of microgravity. The effect of a short-term spaceflight (current space missions) was until now poorly understood. The SOL muscle weight of rats placed in orbit for 7 days aboard Cosmos 1667, compared with vivarium 2 was decreased by 23%, whereas atrophy of the EDL muscle reached 11%. Muscle weight decrements during Cosmos 1667 were similar to those reported during the Spacelab- shuttle flight where the mass of the SOL and EDL muscles was decreased by 36 and 16%, respectively (10, 16). They confirmed the greater atrophy occurring in the SOL muscle after 1 wk of hindlimb tail suspension (37%) (5,6) or harness suspension (29%) (18,19). However, the loss of EDL mass was greater than after 1 wk of Morey’s tail suspension model, since it reached only 4% in the latter case (5). In SOL microgravity induced a slight tendency to a decreased type I fiber percent distribution (11%) counterbalanced by a large significant increase in type IIa fibers. We had only four SOL muscles at our disposal to perform the study, and this may account for the nonsignificant decrease of type I fiber percentage. The effects of the present flight seem to have occurred earlier than those of the tail suspension technique. Indeed, type IIa fiber distribution was significantly increased only after 2 wk of hindlimb suspension (5). After 1 wk a decrease in the percent distribution of type I fibers (85.5 t 2.4 vs. 73.8 $- 2.1%) had b een evidenced, whereas intermediate
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WEIGHTLESSNESS
50
AND MUSCLE
ATROPHY
FIG. 1. Cross sections of rat soleus muscle stained for myofibrillar ATPase activity after preincubation at pH 4.4. A: vivarium 2 group. I, type I muscle fiber; Int I, intermediate type I muscle fiber; IIa, type IIa muscle fiber. B: decrease in percentage and cross-sectional area of type I fibers with concomitant significant increase in percentage of type IIa fibers in flight groups.
TABLE
2. Influence of weightlessness on percent distribution
of fiber type in SOL and EDL % Distribution
SOL (n = 4) I
Int I
EDL (n = 7) IIa
I
II
Vivarium 1 85.5k3.4 4.4k1.6 lO.lk3.9 9.5kO.7 Synchronous 84.5k3.4 8.Ok3.1 7.5f1.9 9.3*0.4* Vivarium 2 88.5k1.7 7.5k1.2 4.OkO.6 lO.Ozb1.7 Flight 78.5k3.4 7.9k3.1 13.6f3.7t 5.3+0.7t Values are means + SE. SOL, soleus; EDL, extensor digitorum longus. Significantly t between vivarium 2 and flight groups. TABLE
3. LDH,
CS, and HAD activities and capillarization
22.321.6 15.9f2.9 19.8k3.3 16.1k2.1 different: *between
Int II
In,
3.6f1.7 64.6kO.8 5.1kl.O 68.Ok3.1 5.2f1.6 65.0k4.6 4.6k1.5 71.9k2.1 synchronous and flight groups,
in SOL and EDL Group
Vivarium 1 LDH, pmol . min-’ . g-’ CS, pm01 . min-’ +g-’ HAD, pmol . min-’ . g-i CD, capillaries/mm* Capillaries/fiber
124.Ok7.0 37.2k3.1 13.2k1.6 842.Ok156.0 2.7eO.4
Synchronous SOL (n = 4) 131.0f19.0 34.5k1.9 ll.Ok2.0 928.0f114.0 2.9f0.3
Vivarium 2
Flight
140.2f17.0 34.6k4.0 12.0+1.5 994.O-c81.0 2.9fO.l
llO.Ozk8.0 35.222.0 12.1kl.O 1,161.0f220.0 2.5f0.3
EDL (n = 7) LDH, rmol . min-’ .g-’ 505.Ok39.0 53o.ozk44.0 546.Ok28.0 528.Ok45.0 CS, pm01 . min-’ . g-’ 34.2zk2.9 30.6f2.5 33.Ok1.3 29.5f2.1 HAD, Fmol .min-’ . g-’ 6.9f0.8 6.5+0.4 7.0f0.8 5.1+0.5* Values are means + SE, n, no. of measurements. LDH, lactate dehydrogenase; CS, citrate synthase; HAD, 3-hydroxyacyl-CoA dehydrogenase; CD, capillary density; SOL, soleus; EDL, extensor digitorum longus. * Significantly different between vivarium 2 and flight groups.
type I fiber distribution was doubled (8.5 -C 1.9 vs. 17.1 -e 1.7%). Type IIa fiber distribution remained unchanged (5.9 + 1.2 vs. 7.3 -I 1.5%). In EDL, during the COSMOS 1667 flight, microgravity induced a reduction of type I fiber from 10 to 5%. Takacs et al. (27) had also observed an increase in the quantity of myosin light chain-3 subunits in the SOL and EDL muscles of rats exposed to weightlessness for 18.5 days (Cosmos 1129). Martin et al. (16) reported an increase in the percentage of dark
ATPase fibers in Spacelab- flight muscles with a predominance of light ATPase fibers. According to our previous experiments (5), the slow-twitch type I fiber becomes a hybrid fiber like the intermediate fiber, representing transitional stages within the steps of the major fiber types (5). The reports of Fitts et al. (8) and Reiser et al. (23) also suggested that the increase in the mean maximal velocity of SOL muscle shortening after hindlimb suspension was associated with an increased expres-
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WEIGHTLESSNESS
AND MUSCLE
sion of a second faster migrating isozyme of myosin, the amount of fast-type myosin heavy chain increasing with relatively small changes in the myosin light chain composition. The findings of Templeton et al. (29) are consistent with our earlier results, indicating that chronic reduction of gravitational load preserves total fiber number and induces a shift in fiber types rather than simultaneous type I fiber loss and type II fiber synthesis, according to the absence or presence of embryonic or neonatal myosin. Steffen and Musacchia (26) also suggested that unaltered DNA contents after a short-term exposure to weightlessness indicated no loss of muscle fibers. After 7 days of microgravity (Cosmos 1667), neither mean capillary density nor capillaries per fiber were affected in SOL or EDL. These data are at variance with those of Musacchia et al. (20), who reported a significant increased density of capillaries (50%) in SOL muscles submitted to 7 days of weightlessness (Spacelab-3). In an earlier experiment, we showed a decrease of 46% of capillaries per fiber after hindlimb suspension but it was after a longer period (5 wk). These modifications of capillary density (20) or capillaries per fiber (5) raise the question of a potential alteration in blood flow to atrophied muscles as suggested by Musacchia et al. (20). No alteration in functional capacity of rat muscles was noted after the Cosmos 1667 flight except in EDL, where a decrease in HAD activity (27%) was observed. In SOL, the absence of reductions in both CS and HAD activities, after 1 wk of weightlessness, is in agreement with our previous observations (5). Indeed, 5 wk of hindlimb suspension were necessary to induce a decrease in these activities (23 and 39%, respectively). This is in contrast with the findings of Fell et al. (7), who observed a reduction in CS activity after 1 wk of harness suspension. In SOL and EDL, the nonaltered LDH activity was identical to that observed after 1 wk of hypokinesiahypodynamia (5). However, after a longer flight (21.5 days, Cosmos 605), Portugalov and Petrova (22) reported alterations in the spectrum of LDH isozymes in favor of an increased glycolytic process. With respect to free fatty acid oxidation, the decrease in HAD activity observed in EDL was discordant with results reported after 1 wk of suspension (5) but was confirmed after 12.5 days for the Cosmos 1887 flight (unpublished data). A maintenance of oxidative enzymatic activity (CS and HAD) had been observed after 7 days for Cosmos 1667. These data are similar to those demonstrated in SOL and EDL muscles by Martin et al. (16) from the Spacelab 3 mission. An interesting point was advanced in their study. To explain the absence of variation in succinic dehydrogenase (SDH) activity, they suggested that the total amount of the oxidative enzyme per fiber (product of cross-sectional area and SDH activity) should be determined. Thus they took into account that a preferential loss of muscle volume (and probably contractile protein) relative to metabolic enzymes may have occurred. Martin et al. (16) argued that their results (increased SDH activity while the total amount of the SDH enzyme per fiber decreased by 21%) point to an interaction between the rate of synthesis and degradation of specific proteins and the
ATROPHY
51
control of cell volume after microgravity. What determines the differential responses of slowand fast-twitch fibers to weightlessness remains an unresolved problem. Martin et al. (16) suggested that the decrease in mechanical loading of muscle may have a more important role than the total amount of electromyographic activity per day because the SOL during suspension regains its normal pattern of activity within 7 days. Furthermore, the role of hormones in the development of muscular atrophy and the slow-to-fast alterations in skeletal muscle fibers should not be underestimated. For example, the growth of fast-twitch fibers is regulated by insulin (3 1), triiodothyronine stimulates the growth of both slow- and fast-twitch muscles, and @adrenergic agonists like clenbuterol (31) cause hypertrophy of fast-twitch fibers in SOL and EDL muscles accompanied by slow-to-fast fiber conversion. A higher concentration of corticosterone and a lower concentration of thyroxine in blood was reported after flight, showing the emergence of an acute gravitational stress after flight. In conclusion, histochemical changes show that muscular atrophy is accompanied by an increased expression of fast-type myosin in the slow-type I fiber in SOL. No variation in capillarization or in the three enzyme activities was observed except a decrease in HAD for the EDL. Similar tendencies in groups S and F indicate that muscular alterations reflect the effect of weightlessness rather than confinement. The present observations are consistent with those obtained in the tail suspension model, suggesting the interest of ground-based simulations to study the effects of microgravity on muscle. The small structural and functional muscular adaptations reported after short-term exposure to weightlessness demonstrate the need to study muscular adaptations during a flight of longer duration. The authors are grateful to the staff of the Institute of Biomedical Problems, especially 0. G. Gazenko and Dr. E. A. Ilyin. They thank Dr. V. S. Oganov and V. S. Skuratova for providing the muscle samples used in these studies. They also thank Dr. M. J. Carew for reviewing the English manuscript. This work was supported by a grant from Centre National d’Etudes Spatiales. Address for reprint requests: D. Desplanches, UA CNRS 1341, Laboratorie de Physiologie, Lyon Grange-Blanche Universitit, 69373 Lyon Cedex 08, France. Received 21 February 1989; accepted in final form 30 August 1989. REFERENCES Capillary supply of the quad1. ANDERSEN, P., AND J. HENRIKSSON. riceps femoris muscle of man: adaptive response to exercise. J. Physiol. Lord 270: 667-690, 1977. 2. BOOTH, F. W., AND J. R. KELSO. Effect of hindlimb immobilization on contractile and histochemical properties of skeletal muscle. Pfhegers Arch. 342: 231-238, 1973. 3. BROOKE, M. H., AND K. K. KAISER. Muscle fiber types: how many and what kind? Arch. Neural. 23: 369-379, 1970. 4. CHUI, L. A., AND K. R. CASTLEMAN. Morphometric analysis of rat muscle fibers following spaceflight and hypogravity. Pkysiologist 23, Suppl.: S576-S578,1980. 5. DESPLANCHES, D., M. H. MAYET, B. SEMPORE, AND R. FLANDROIS. Structural and functional responses to prolonged hindlimb suspension in rat muscle. J. Appl. Physiol. 63: 558-563, 1987. 6. DESPLANCHES, D., M. H. MAYET, B. SEMPORE, J. FRUTOSO, AND R. FLANDROIS. Effect of spontaneous recovery or retraining after
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