~~
~~~~~~
The aim of this study was to investigate the contractile protein characteristics after 5-day (Cosmos 1514) and 7-day (Cosmos 1667) spaceflights. The experiments were performed on skinned fibers from the soleus, gastrocnemius lateralis, and plantaris muscles isolated from Wistar rats. A reduction in fiber diameter might explain the decrease in the maximal tension in the soleus, whereas this tension was unaltered in the gastrocnemius and the plantaris. Moreover the calcium sensitivity of the myofilament appeared modified in the soleus and in the gastrocnemius: The tension/pCa relationships were shifted toward higher calcium concentrations, indicating a decrease in the apparent calcium binding constant of the tropinin C. The tension/pCa relationship appeared unaltered in the plantaris after spaceflight. Finally, the studies of the time to reach a steady tension indicated an increase in the rate of force development in the soleus and, on the contrary, a slowing down in the plantaris. No change in the gastrocnemius was found. The results were analyzed with references to the different muscle functions in disuse atrophy. Key words: spaceflight skinned fiber atrophy myofilament calcium sensitivity MUSCLE 81 NERVE 14~70-78 1991
EFFECTS OF SHORT SPACEFLIGHTS ON MECHANICAL CHARACTERISTICS OF RAT MUSCLES XAVIER HOLY and YVONNE MOUNIER
Spaceflights and consecutive microgravity have been shown to induce many biological disorders in humans and aniniak2.'4 -The muscu~oskeletalsystem was especially involved. Decrease in body mass, leg volume, and muscle mass have been described.'"."' A decrease in muscle fiber diameter, modification of composition, and preferential reduction of the slow twitch fibers have been repor~ed."'.'~ It is well known that the contractile machinery is switched on by calcium ions. T h e troponin complex binds calcium and regulates the availability of potential cross-bridge binding sites by influencing the thin filament structure. Finally, myosin
ATYase activity occurs and tension develops. Therefore, the amount of tension developed by a muscle fiber can vary according to the calcium concentration in the myoplasm. To investigate directly the contractile mechanism we used skinned fibers. A skinned fiber is defined as a preparation in which sarcolemma disruption permits direct access to myofilaments and control of their sensitivity to calcium ion^.^,^'',^^ In the present study, the effect of microgravity on the contractile properties of skinned muscle fibers was examined. Maximal tensions, myofilament calcium sensitivity, and the rate of force development were studied.
From the Laboratoire de Physiologie des Structures Contractiles, Universite des Sciences et Techniques de Lille, Villeneuve d'Ascq, France
Animals and Muscles.
'
MATERIALS AND METHODS
Acknowledgments: This work was supported by grants from the "Centre National d'Etudes Spatiales." The authors thank the Institute of Biomedical Problems (Moscow) for providing the biological material from the two biocosmos and they are grateful to Drs Oganov, Skuratova, and Murashko for their scientific cooperation and work on the landing area in USSR Address reprint requests to Y. Mounler. Laboratoire de Physiologie des Structures Contractiles, SN4. Universite des Sciences et Techniques de Lille, F-59655 Villeneuve d'Ascq. Cedex. France. Accepted for publication December 25, 1989. CCC 0148-639X/91/01070-09 $04 00 0 1991 John Wiley & Sons, lnc
70
Muscle Properties after Weightlessness
Experiments were performed on three muscles from Wistar rats, soleus, plantaris, and gastrocnemius lateralis. T h e soleus is a slow-twitch type muscle containing 87% slow oxydative (SO) fibers, 1:3% fast oxydative glycolytic (FOG) fibers, and 0% fast glycolytic (FG) fibers while the plantaris and the gastrocnemius lateralis are f-ast-twitch muscles containing, respectively, 9% and 7% SO fibers, 50% and 28% FOG fibers, and 41% and 65% FG fibers.' Moreover, the soleus and the gastrocnemius must be consid-
MUSCLE & NERVE
January 1991
ered as muscles involved in maintaining posture to a greater extent than the plantaris muscle. In biocosmos 1514, five female rats were orbited for a 5-day spaceflight (12/14/1983 to 12/19/ 1983). In biocosmos 1667, 7 male rats were placed in orbit for 7 days (07/10/1985 to 07/17/1985). Individual cages were used for flight animals (F). Each rat received a pastelike diet 4 times per day every 6 hours and water ad libitum. T h e temperature was about 24°C and the light-dark cycle (121 12 hours) was regulated. Synchronous animals (S), so called because they received flight simulation in a spacecraft mock-up, were considered as the control group. (S and F were used as international symbols by the different participants in the experiments.) Indeed, they were submitted to the same stresses as the flight group except weightlessness (individual cages, food, water, lighting, temperature, launch and reentry stresses such as vibration, acceleration, impact shock, and noise). Each animal was weighed after and before the flights and the ground simulated experiments. The mean weights of the synchronous animals increased by 10% during the 5-day and 7-day simulations, reaching weights equal to 359.0 k 3.2 g (n = 5) and 333.8 5 5.9 g (n = 7) respectively. T h e mean weights of the flight animals increased by 2% during both flights reaching 293.4 t 6.5 g ( n = 5, flight 1514) and 303.8 17.4 g ( n = 7, flight 1667) when the animals were sacrificed. Moreover the mass of the 3 muscles significantly decreased after the flights (Table 1). T h e small body weight increase during the flights could be attributed to the fact that the whole muscle mass declines, at least to a certain extent.
*
Dissection and Skinning. Animals were killed by decapitation 6 h after landing or at the end of the simulated experiment. Muscles were removed and biopsies (15-20 mm long, 5 mm in diameter)
were immediately taken and immersed in a skinning solution.Y2Biopsies were stored at -20°C in a 50% (vol/vol) glycerol-skinning solution. They remained intact and kept their physiological properties for about 5 weeks after the removal of the muscles. On the day of the experiment, a single-skinned fiber was isolated from a biopsy, transferred, and mounted in the experimental chamber. One end was held by a fixed forceps and the other end by an isometric transducer (Kulite, model BG 10, sensitivity equal to 0.70 V/g). Before the experiment, the fiber was bathed twice for 10 min into the relaxing solution containing 2% (w/v) BRIJ 58 (polyoxyethylene 20 cetyl ether). T h e detergent-treated preparation had no more functional sarcoplasmic reticulum (SR) but its actomyosin system remained intact.'3 T h e length of all the fiber segments used for the experiments was limited to about 900 p,m. The mounted fiber was viewed through a highmagnification binocular and measurement of the fiber diameter was made using a reticule ( x 80) assuming that the cross-section was circular. Fibers with a very elliptical appearance were not used in order to minimize the approximation in the crosssection area measurements. Using the diffraction of a He-Ne laser beam, the overall segment length was adjusted to achieve an average striation spacing of about 2.6 p,m. This sarcomere length corresponded to a fiber length stretched to 120% of the rest length. This length was used to allow maximal isometric tension. It was then regularly controlled and readjusted when necessary during the experiment while the fiber remained in the relaxing solution. The experimental procedure was defined as follows: T h e fiber was washed and a tension (P) was elicited by a given pCa solution (pCa = -log [Ca]). When the steady level of the contraction was Force Measurement and Recording.
Table 1. Muscle mass after the 1514 (5-day) and 1667 (7-day) spaceflights. Soleus
Gastrocnemius
Plantaris
S (5-day) F (5-day)
131.62 ? 13.61 108.61 ? 4.04*
731.62 2 23.91 556.23 5 39.28*
278.02 2 16.85 229.63 ? 13.61*
S (7-day)
172.43 ? 6.01 122.42 ? 5.01*
1085.71 2 33.08 982.85 ? 38.15*
325.42 2 15.65 281.00 2 7.47'
F (7-day)
Data expressed in milligrams, * indfcates a significant difference (P < 0 05) between synchronous and fhght rats Animals for S and F groups were obtafned from the same initial group of rats before each flight
Muscle Properties after Weightlessness
MUSCLE & NERVE
January 1991
71
reached, the tiber was relaxed by the relaxing solution. T h e n , the fiber was washed and a maximal tension (Po) was elicited. T h e solution used for this purpose was a pCa 4.8 solution. lndeed higher calcium concentrations (lower pCa) did not induce greater tensions (see Fig. 2). This proced u r e permitted the calculation of the relative tension (P/Po). T h e n , a new cycle could be started using another pCa solution. All experiments were performed at 17 +- 1°C. All the data are presented as means +- SEM (standard error of mean). T h e statistical significance of the differences between means was determined using the Student's t test. Differences at or above the 95% confidence level were considered significant. T h e components of all the solutions were added in concentrations calculated by program 3 of Fabiato and Fabiato" in order to keep the ionic strength at 200 M.T h e stability constants of Orentlicher et a12' were used in the calculations: KCaEGrA 1.919 x 10" M-', KCaA-l.I,5.0 X 10' M - ' , KMvlgE(;I-A 40 M - ' and KMvlgA.I.I, 1.0 X lo4 M-'. T h e p H of all the solutions was adjusted to 7.00 0.02. Skinning solution: A T P (2.5 nliM), M O P S (Morpholinopropane sulfonic acid, 10 r n k f ) , potassium propionate (170 mM), magnesium acetate (2.5 mM), and K,EGTA (5 M). Storuge .solution: Fifty percent skinning solution and 50% glycerol.
solutions.
Solutions used f o r the experirnerit procedure.: Wnsh .solution (W): A T P (2.5 mM), MOPS (10 mM), potassium propionate (185 mM), and magnesium acetate (2.5 mM). Ralnxing solution (R): Similar to the skinning solution. ~ C solutioris: Q Wash solution (W) + various concentrations of' free Cay+ (from CaCO:,) buffered with EGI-A and added in proportions to obtain the different pCa values. Brij .solution: Kelaxing solution (K) + 2% Brij 58. RESULTS Fiber Diameter. T h e diameter of the skinned fibers was measured before each experiment. T h e data are summarized in Table 2. T h e diameter of the soleus muscle fibers decreased b y 14% after the 7-day spaceflight. 'The gastrocnemius muscle also exhibited a small significant decrease in fiber size after the 5-day spaceflight although the variations were not significantly different from the 7day spaceflight. T h e plantaris muscles showed no significant modification of fiber diameter following both flights.
*
-1he maximal tensions (Po) were obtained by application of a pCa 4.8 solution, a calcium concentration large enough to saturate all the sites of T n C ; and thus to create the maximal number of cross-bridges. Typical records from the S muscles were given in Figure 1. They showed that the Inaximal tensions were smaller Maximal Tension.
Table 2. Muscle fiber characteristics after the 1514 (5-day) and 1667 (7-day) spaceflights. Maximal tension (Po)
Muscle
n
Diameter (Pm)
Soleus S (7-day) F (7-day)
29 16
78 02 5 2 68 67 18 2 3 47**
5 6 1 2 i 326 40 13 f 3 32**
121 f 007 1 34 5 0 4 9
Gastrocnemius lateralis S (5-day) F (5-day) S (7-day) F (7-day)
26 16 13 12
85 15 i 2 02 78 60 5 2 64* 88 30 t 4 49 8315t293
88 26 6841 87 30 77 39
i 5 91
155 i 017 1 2 3 k 013 1 45 2 0 08 1 41 2 0 0 7
Plantaris S (5-day) F (5-day) S (7-day) F (7 day)
12 12 12 29
65 63 5 3 83 58 85 2 2 99 70 00 f 2 04 70 03 5 3 20
61 85 k 6 13 57 53 4 39 62 01 285 64 70 -t 4 15
1 8 7 5 013 2 13 2 0 18 1645012 1705013
mg
5
kgicm
6 52
t 5 13*
t 4 49
*
'
' P < 0 05 **P < 0 01
72
Muscle Properties after Weightlessness
MUSCLE . 8 NERVE
January 1991
S
F
I
A
1
SOLEUS
cross-sectional area and expressed in kg/cm2. The tensions per surface unit were not significantly changed either in the slow soleus muscle or in the fast gastrocnemius and plantaris muscles. This meant that atrophy was the main cause of the force decrease.
T h e relationships between the relative tension (P/P,) and the calcium Concentration expressed in pCa values were established for the soleus, the gastrocnemius and the plantaris. They were called T/pCa relationships with -I‘defined as the P/P,, ratio. T h e results obtained for the 1667 spaceflight are illustrated in Figure 2. T h e curves were established from the Hill equation5: PIP,, = [Ca]” / (K” + [Ca]”) where n is the Hill coefficient and K is the apparent dissociation constant. From these curves, different parameters were determined. T h e Ca2+ threshold for activation of the contractile proteins was defined as the lowest Ca2+concentration required to obtain the development of a tension. Then, the position of the ‘T/pCa curves could be characterized by the pCa,, value which corresponded to the midpoint of the ‘l’/pCa curve, ie, the pCa value at which half-maximal tension was elicited. This parameter indicated the apparent Cay+ affinity. Moreover Hill plots (Fig. 3 ) were used to determine the steepness of the curves. From each Hill plot, two Hill coefficients were measured: n , for the slope of the curve when PIP,, was more than 0.50 and n2 for the slope when P/P, was less than 0.50. Finally, all the results are summarized in Table 3. For the soleus muscle, the activation threshold was higher after the 1667 spaceflight. It was respectively equal to pCa 6.40 and pCa 6.60 for flight (F) and synchronous (S) animals. This meant that after flight the fibers started to contract with a higher calcium concentration when compared to control. T h e pCa,,, values were decreased from 5.90 (S) to 5.75 (F). In Figure 3 it appeared that n I and n2 parameters were higher after the 1667 spaceflight (see Table 3 ) . T h e ‘T/pCa relationship established for the gastrocnemius showed a slightly higher threshold f o r calcium activation (Table 3 ) . ‘l’his threshold was equal to 6.30 (S) and 6.20 (F) after the 7-day spaceflight (Fig. 2U). As described for the soleus, the curve was shifted toward lower pCa values and the pCa.,[) values decreased from 5.97 (S) to 5.87 (F) in the 7-day flight. A shift by 0.15 pCa unit was measured after the 5-day flight. The n I values
TensionlpCa Relationships.
4.8
R
4.8
R
FIGURE 1. Records of maximal tensions induced by a pCa 4.8 solution on single-skinned fibers of soleus, gastrocnemius lateralis, and plantaris muscles for synchronous (S) and flight (F) animals (flight 1667). When the steady maximal tension was reached, the fiber was relaxed by the “R” solution. The fast transient peaks on the records and the transient displacements of the base line are produced by the chgnges of the solutions. The reference level used for the amplitude measurements is shown by a dashed line just before the record.
for the soleus muscle after spaceflights whereas they remained unchanged for the gastrocnemius and plantaris muscles. T h e results of both flights are summarized in Table 2. The soleus muscle fibers exhibited a clear decline in the absolute maximal tension expressed in mg (up to 28% after the 1667 biocosmos). T h e gastrocnemius showed a significant modification when the tension was expressed in milligrams after the 1514 spaceflight while a less important decrease appeared after the 1667 spaceflight (not significantly different from the controls). In the plantaris muscle, no change was observed after both spaceflights. All these results suggested that atrophy particularly occurred in the slow muscle. Since it is well known that tension is dependent on the fiber size, the maximal tensions were also calculated in relation to the
Muscle Properties after Weightlessness
MUSCLE & NERVE
January 1991
73
-
n 0
100-
50
Table 3. Parameters of the TlpCa relationships
A
.
Activation threshold (APW
F
4 W
ApCa,,
a
0
-
"1
B -
n2
0
a 100- B
z 0 u)
bI-
Sol GL PL Sol GL PL Sol GL PL Sol GL PL
ControllFlight 1514
ControllFlight 1667
-
0 20 0 10 0 0 15 0 10 0 1 6611 81 2 7712 88 2 7012 70 3 0013 14 5 8814 54 4 9014 90
0.10 0 -
0.15 0 -
2.6612.40 2.6612.66 -
5.7114.00
5.0015.00
S o l , G L , and P L designed respectively the soleus. the gastrocnemius lateralis, and the plantans muscles Results for the activation threshold and the pCa,, parameters were expressed in terms of differences between control (synchronous) and flight values 0 indicated no difference Dash indicates no result n, and np were the slopes of the fitted line above and below 50% of PiPo respectively
W
2 -I
W
II:
6.4
5.6
4.8
change occurred in the tension/pCa relationship. T h e n , and n2 parameters remained unchanged.
z
8z
50.
W
I-
PLANTARIS
W
2
4 w
II:
FIGURE 2. Relative tensionlpca relationships in soleus (A), gastrocnemius lateralis (B), and plantaris (C) muscles for synchronous (filled circles) and flight (open circles) fibers. Curves were drawn through the experimental points according to the Hill equation using n, and n2 Hill parameters (see text). Values are means 2 SEM. The number of fibers in each curve was the same as listed in Table 2 for the 7-day results.
were only slightly modified -respectively decreased o r increased after the 15 14 and the 1667 spaceflights ('Table 3). This slight modification indicated that this part of the relationship (>pCa,,,) was not clearly changed by the flights. On the contrary, the n2 values were clearly decreased for both flights. T h u s the influence of the spaceflights seemed more apparent when the fi-ee Ca2+ concentration was relatively low. T h e plantaris muscle was unaffected by the 2 spaceflights. Similar thresholds equal to 6.30, similar pCa,,, values equal to 5.85 were measured both in the synchronous and flight animals. N o
74
Muscle Properties after Weightlessness
An attempt to compare the tension kinetics in synchronous and flight animals was made by measuring the time to achieve the maximum tension level (t,,,',,). We were aware that this parameter might be an imperfect indicator of muscle fiber velocity in skinned fiber. However, o u r purpose was only to determine the possible effect of the flights essentially, from a qualitative point of view. l h e data from the three muscles for the 1667 flight (7 days) are summarized in Figure 4. Similar results were obtained after the 1514 flight (5 days). T h e rate of force development observed on the synchronous animals for the three muscles depended on the amplitude of the tensions elicited by the different pCa solutions. T h e higher the pCa value, the lower the tension and the larger the t,,,,,. In synchronous animals, the values of t,,,',, depended on the muscle. For the maximal tensions, t,,,,, varied as follows: plantaris ( 3 sec) < gastrocnemius (6 sec) < soleus (12 sec). These values are in good agreement with those generally proposed in ratskinned fibers.20 In the soleus muscle, the tensions developed faster after spaceflight (Fig. 4A), which was true whatever the initial tension amplitude. In the gastrocnemius, the spaceflight induced a slight decrease in the rate of force development (Fig. 4B). In the plantaris (Fig. 4C) the rate of contraction Rate of Force Development.
MUSCLE & NERVE
January 1991
firmed that a 5 or a 7-day spaceflight was long enough to induce changes in the contractile proteins.
A consistent atrophy appeared in the soleus muscle since we found a significant decline in muscle mass and fiber diameter. A significant decrease in the gastrocnemius and plantaris muscle mass also happened after both flights while in the gastrocnemius the decrease in fiber diameter clearly occurred only after the 1514 flight. So, as generally reported,'"."'3" the atrophy was more pronounced in the slow soleus muscle.
Muscle Atrophy.
-3'. . 7.2
-
*
6.4
.
-
-
5.6
-
.
'
4.8
B
7
s
a -3l
.
7.2
.
1
6.4
5.6
4.8
C n
l i
-* 7.2
6.4
5.6
0 6.4
5.6
4.8
0' 6.4
5.6
4.8
5.6
4.8
4.8
pCa FIGURE 3. Hill plots of the mean values of the pCa-force data for fibers from the soleus (A), the gastrocnemius lateralis (B), and the plantaris (C) muscles (number of fibers as in Fig. 2 in each group). Synchronous fibers (filled circles) and flight fibers (open circles).
I
always decreased significantly after the spaceflights. DISCUSSION
Spaceflights have been well known to induce muscle a t r o p ~ y ~ ~ . 7 . 1 : ~ , ~ 0and . 2 1 most of the results were generally obtaiqed after 18- to 20-day spaceflights. A major point emphasized in this article is that space atrophy and physiological changes happened after spaceflights as short as 5 or 7 days. Recently, biochemical changes after a 7-day flight (SL3) have been described." Moreover on the hindlimb suspended rat, the half-time of the degradation rate constant in the soleus was 4.1 days for the myofibril proteins and 3.6 days for the slow myosin content.") Therefore, our results con-
Muscle Properties after Weightlessness
0' 6.4
PCa FIGURE 4. Relationships between tmaX the time to reach the maximum level of the tension and pCa in soleus (A), gastrocnemius lateralis (B), and plantaris (C) muscles for synchronous (filled circles) and flight (open circles) fibers. Values are means +. SEM. Number of fibers as in Figure 2.
MUSCLE & NERVE
January 1991
75
The changes in the fast muscles were less marked. However we must point out that the flights had more marked effects on the gastrocnemius, a fast muscle with an antigravitational function than on the plantaris, a fast-twitch muscle used in ankle movement. T h e decline in fiber size has usually been described in various disuse atrophy situations such as immobilization (casting, bed rest, or water immersion), denervation, and tenotomy. It was generally explained by a decrease in the content of myofibril proteins. The main explanation might be a reduction in the rate of protein synthesis in conjunction with an elevation in the rate of protein degradation." When the muscle loss was not associated with a fiber diameter decline, it was attributed primarily to a loss of muscle tissue Our results showed changes in the maximal tension dependent on muscle type. A very significant decrease in P,, expressed in milligrams appeared in the soleus as already described on glycerinated fibers."',21 T h e decrease was more variable for the gastrocnemius (significant for the 1514 flight and not significant for the 1667 one) and the fiber type heterogeneity of this muscle might be responsible for the difference between the two flights. N o modification occurred in the plantaris, a fast muscle. This was already described for the EDI, (extensor digitorum longus), another fast muscle after biocosmos 936 (18.5 These authors even reported an increase in EDL tension after another biocosmos (1 129 flight, 18.5 days). Moreover the decrease in absolute values of P,, disappeared when the force was expressed per cross-sectional area. Since a decrease of maximal tension was usually related to a decrease of the maximal cross-bridge number between actiri and myosin, our result indicated that a decrease in the number of cross-bridges was more probable than a change in density or a change of force per cross-bridge. S o , the decrease of muscle strength after a spaceflight could result from muscle atrophy. T h e data from the TlpCa curves indicated that the value of the Ca2+ threshold for activation in the soleus and in the gastrocnemius was higher after spaceflights. So, the sensitivity of' the myofilaments for Ca2+ was decreased. T h e apparent Ca2' threshold for activation was well known to be higher for the fast fibers,11,2(i the highest threshold being obtained for intermediate fibers." 'l'herefore, our result suggested that the slow soleus muscle appeared to
Ca2+ Affinity of the Contractile Proteins.
76
Muscle Properties after Weightlessness
get Cay+ threshold activation characteristics that resembled more those of a fast muscle. On the other hand, the initially fast gastrocnemius lateralis muscle might modify its characteristics towards those of an intermediate muscle. No significant change in the calcium threshold for the plantaris was observed. Moreover, we described a shift of the tension/ pCa relationships of the soleus and gastrocnemius toward lower pCa values while that of the plantaris remained unchanged. T h e position of the T/ pCa curve was generally assumed to be directed by the Ca2+ binding properties. Indeed, an increase in the isometric tension in skinned fibers was presumed to parallel the binding of Ca'+ to regulatory sites on troponin C.I6 Thus, the shifts toward the right of the T/pCa curves for soleus and gastrocnemius after flights were assumed to parallel the decreases in the apparent Ca2+ affinity of the tropinin C, illustrated by the decrease of pCa,,, values. Another result was the Hill coefficient measurements, n , and n2, representative of the steepness of the T/pCa relationships. Two coefficients were necessary to obtain the best fit between the theoretical curve and our experimental values: n , for the curve above pCa,,, and n2 for the curve below pCa,,,. A similar analysis was already used by different authors. When changes occurred, they were more obvious for the n2 coefficient. In most current models of muscle activation, the value of the Hill coefficients reflected the cooperativity among Ca2+ binding sites along the thin filament. So, the increase observed in the soleus, especially for the n2 value might be explained by a greater cooperativity. Then, the soleus muscle seemed to react more like a fast muscle. In the gastrocnemius the changes of the n , values were not homogeneous (slight decrease after the 1514 flight and slight increase after the 1667 flight) while the n2 values were clearly decreased after both flights. This should indicate a lower cooperativity of the Ca2+ binding sites in the gastrocnemius muscle more characteristic of a slower muscle. In plantaris, n , and n2 values remained unchanged after both flights. Experiments of partial substitution with troponin C from different types".'7,''' clearly showed that the position and steepness of the T/pCa curves were strongly dependent on the type of troponin C present in the muscle. This indicated that troponin C played a direct role in the cooperative mechanism.5 However, our values of n,, especially in the fast muscles as well as the nE3values
MUSCLE & NERVE
January 1991
previously obtained by Brandt et a14 on the rabbit psoas ( n Hfrom 5.0 to 5.6) were too large to be explained by a control mechanism governed exclusively by
~~~~~~
The aim of this study was to investigate the contractile protein characteristics after 5-day (Cosmos 1514) and 7-day (Cosmos 1667) spaceflights. The experiments were performed on skinned fibers from the soleus, gastrocnemius lateralis, and plantaris muscles isolated from Wistar rats. A reduction in fiber diameter might explain the decrease in the maximal tension in the soleus, whereas this tension was unaltered in the gastrocnemius and the plantaris. Moreover the calcium sensitivity of the myofilament appeared modified in the soleus and in the gastrocnemius: The tension/pCa relationships were shifted toward higher calcium concentrations, indicating a decrease in the apparent calcium binding constant of the tropinin C. The tension/pCa relationship appeared unaltered in the plantaris after spaceflight. Finally, the studies of the time to reach a steady tension indicated an increase in the rate of force development in the soleus and, on the contrary, a slowing down in the plantaris. No change in the gastrocnemius was found. The results were analyzed with references to the different muscle functions in disuse atrophy. Key words: spaceflight skinned fiber atrophy myofilament calcium sensitivity MUSCLE 81 NERVE 14~70-78 1991
EFFECTS OF SHORT SPACEFLIGHTS ON MECHANICAL CHARACTERISTICS OF RAT MUSCLES XAVIER HOLY and YVONNE MOUNIER
Spaceflights and consecutive microgravity have been shown to induce many biological disorders in humans and aniniak2.'4 -The muscu~oskeletalsystem was especially involved. Decrease in body mass, leg volume, and muscle mass have been described.'"."' A decrease in muscle fiber diameter, modification of composition, and preferential reduction of the slow twitch fibers have been repor~ed."'.'~ It is well known that the contractile machinery is switched on by calcium ions. T h e troponin complex binds calcium and regulates the availability of potential cross-bridge binding sites by influencing the thin filament structure. Finally, myosin
ATYase activity occurs and tension develops. Therefore, the amount of tension developed by a muscle fiber can vary according to the calcium concentration in the myoplasm. To investigate directly the contractile mechanism we used skinned fibers. A skinned fiber is defined as a preparation in which sarcolemma disruption permits direct access to myofilaments and control of their sensitivity to calcium ion^.^,^'',^^ In the present study, the effect of microgravity on the contractile properties of skinned muscle fibers was examined. Maximal tensions, myofilament calcium sensitivity, and the rate of force development were studied.
From the Laboratoire de Physiologie des Structures Contractiles, Universite des Sciences et Techniques de Lille, Villeneuve d'Ascq, France
Animals and Muscles.
'
MATERIALS AND METHODS
Acknowledgments: This work was supported by grants from the "Centre National d'Etudes Spatiales." The authors thank the Institute of Biomedical Problems (Moscow) for providing the biological material from the two biocosmos and they are grateful to Drs Oganov, Skuratova, and Murashko for their scientific cooperation and work on the landing area in USSR Address reprint requests to Y. Mounler. Laboratoire de Physiologie des Structures Contractiles, SN4. Universite des Sciences et Techniques de Lille, F-59655 Villeneuve d'Ascq. Cedex. France. Accepted for publication December 25, 1989. CCC 0148-639X/91/01070-09 $04 00 0 1991 John Wiley & Sons, lnc
70
Muscle Properties after Weightlessness
Experiments were performed on three muscles from Wistar rats, soleus, plantaris, and gastrocnemius lateralis. T h e soleus is a slow-twitch type muscle containing 87% slow oxydative (SO) fibers, 1:3% fast oxydative glycolytic (FOG) fibers, and 0% fast glycolytic (FG) fibers while the plantaris and the gastrocnemius lateralis are f-ast-twitch muscles containing, respectively, 9% and 7% SO fibers, 50% and 28% FOG fibers, and 41% and 65% FG fibers.' Moreover, the soleus and the gastrocnemius must be consid-
MUSCLE & NERVE
January 1991
ered as muscles involved in maintaining posture to a greater extent than the plantaris muscle. In biocosmos 1514, five female rats were orbited for a 5-day spaceflight (12/14/1983 to 12/19/ 1983). In biocosmos 1667, 7 male rats were placed in orbit for 7 days (07/10/1985 to 07/17/1985). Individual cages were used for flight animals (F). Each rat received a pastelike diet 4 times per day every 6 hours and water ad libitum. T h e temperature was about 24°C and the light-dark cycle (121 12 hours) was regulated. Synchronous animals (S), so called because they received flight simulation in a spacecraft mock-up, were considered as the control group. (S and F were used as international symbols by the different participants in the experiments.) Indeed, they were submitted to the same stresses as the flight group except weightlessness (individual cages, food, water, lighting, temperature, launch and reentry stresses such as vibration, acceleration, impact shock, and noise). Each animal was weighed after and before the flights and the ground simulated experiments. The mean weights of the synchronous animals increased by 10% during the 5-day and 7-day simulations, reaching weights equal to 359.0 k 3.2 g (n = 5) and 333.8 5 5.9 g (n = 7) respectively. T h e mean weights of the flight animals increased by 2% during both flights reaching 293.4 t 6.5 g ( n = 5, flight 1514) and 303.8 17.4 g ( n = 7, flight 1667) when the animals were sacrificed. Moreover the mass of the 3 muscles significantly decreased after the flights (Table 1). T h e small body weight increase during the flights could be attributed to the fact that the whole muscle mass declines, at least to a certain extent.
*
Dissection and Skinning. Animals were killed by decapitation 6 h after landing or at the end of the simulated experiment. Muscles were removed and biopsies (15-20 mm long, 5 mm in diameter)
were immediately taken and immersed in a skinning solution.Y2Biopsies were stored at -20°C in a 50% (vol/vol) glycerol-skinning solution. They remained intact and kept their physiological properties for about 5 weeks after the removal of the muscles. On the day of the experiment, a single-skinned fiber was isolated from a biopsy, transferred, and mounted in the experimental chamber. One end was held by a fixed forceps and the other end by an isometric transducer (Kulite, model BG 10, sensitivity equal to 0.70 V/g). Before the experiment, the fiber was bathed twice for 10 min into the relaxing solution containing 2% (w/v) BRIJ 58 (polyoxyethylene 20 cetyl ether). T h e detergent-treated preparation had no more functional sarcoplasmic reticulum (SR) but its actomyosin system remained intact.'3 T h e length of all the fiber segments used for the experiments was limited to about 900 p,m. The mounted fiber was viewed through a highmagnification binocular and measurement of the fiber diameter was made using a reticule ( x 80) assuming that the cross-section was circular. Fibers with a very elliptical appearance were not used in order to minimize the approximation in the crosssection area measurements. Using the diffraction of a He-Ne laser beam, the overall segment length was adjusted to achieve an average striation spacing of about 2.6 p,m. This sarcomere length corresponded to a fiber length stretched to 120% of the rest length. This length was used to allow maximal isometric tension. It was then regularly controlled and readjusted when necessary during the experiment while the fiber remained in the relaxing solution. The experimental procedure was defined as follows: T h e fiber was washed and a tension (P) was elicited by a given pCa solution (pCa = -log [Ca]). When the steady level of the contraction was Force Measurement and Recording.
Table 1. Muscle mass after the 1514 (5-day) and 1667 (7-day) spaceflights. Soleus
Gastrocnemius
Plantaris
S (5-day) F (5-day)
131.62 ? 13.61 108.61 ? 4.04*
731.62 2 23.91 556.23 5 39.28*
278.02 2 16.85 229.63 ? 13.61*
S (7-day)
172.43 ? 6.01 122.42 ? 5.01*
1085.71 2 33.08 982.85 ? 38.15*
325.42 2 15.65 281.00 2 7.47'
F (7-day)
Data expressed in milligrams, * indfcates a significant difference (P < 0 05) between synchronous and fhght rats Animals for S and F groups were obtafned from the same initial group of rats before each flight
Muscle Properties after Weightlessness
MUSCLE & NERVE
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71
reached, the tiber was relaxed by the relaxing solution. T h e n , the fiber was washed and a maximal tension (Po) was elicited. T h e solution used for this purpose was a pCa 4.8 solution. lndeed higher calcium concentrations (lower pCa) did not induce greater tensions (see Fig. 2). This proced u r e permitted the calculation of the relative tension (P/Po). T h e n , a new cycle could be started using another pCa solution. All experiments were performed at 17 +- 1°C. All the data are presented as means +- SEM (standard error of mean). T h e statistical significance of the differences between means was determined using the Student's t test. Differences at or above the 95% confidence level were considered significant. T h e components of all the solutions were added in concentrations calculated by program 3 of Fabiato and Fabiato" in order to keep the ionic strength at 200 M.T h e stability constants of Orentlicher et a12' were used in the calculations: KCaEGrA 1.919 x 10" M-', KCaA-l.I,5.0 X 10' M - ' , KMvlgE(;I-A 40 M - ' and KMvlgA.I.I, 1.0 X lo4 M-'. T h e p H of all the solutions was adjusted to 7.00 0.02. Skinning solution: A T P (2.5 nliM), M O P S (Morpholinopropane sulfonic acid, 10 r n k f ) , potassium propionate (170 mM), magnesium acetate (2.5 mM), and K,EGTA (5 M). Storuge .solution: Fifty percent skinning solution and 50% glycerol.
solutions.
Solutions used f o r the experirnerit procedure.: Wnsh .solution (W): A T P (2.5 mM), MOPS (10 mM), potassium propionate (185 mM), and magnesium acetate (2.5 mM). Ralnxing solution (R): Similar to the skinning solution. ~ C solutioris: Q Wash solution (W) + various concentrations of' free Cay+ (from CaCO:,) buffered with EGI-A and added in proportions to obtain the different pCa values. Brij .solution: Kelaxing solution (K) + 2% Brij 58. RESULTS Fiber Diameter. T h e diameter of the skinned fibers was measured before each experiment. T h e data are summarized in Table 2. T h e diameter of the soleus muscle fibers decreased b y 14% after the 7-day spaceflight. 'The gastrocnemius muscle also exhibited a small significant decrease in fiber size after the 5-day spaceflight although the variations were not significantly different from the 7day spaceflight. T h e plantaris muscles showed no significant modification of fiber diameter following both flights.
*
-1he maximal tensions (Po) were obtained by application of a pCa 4.8 solution, a calcium concentration large enough to saturate all the sites of T n C ; and thus to create the maximal number of cross-bridges. Typical records from the S muscles were given in Figure 1. They showed that the Inaximal tensions were smaller Maximal Tension.
Table 2. Muscle fiber characteristics after the 1514 (5-day) and 1667 (7-day) spaceflights. Maximal tension (Po)
Muscle
n
Diameter (Pm)
Soleus S (7-day) F (7-day)
29 16
78 02 5 2 68 67 18 2 3 47**
5 6 1 2 i 326 40 13 f 3 32**
121 f 007 1 34 5 0 4 9
Gastrocnemius lateralis S (5-day) F (5-day) S (7-day) F (7-day)
26 16 13 12
85 15 i 2 02 78 60 5 2 64* 88 30 t 4 49 8315t293
88 26 6841 87 30 77 39
i 5 91
155 i 017 1 2 3 k 013 1 45 2 0 08 1 41 2 0 0 7
Plantaris S (5-day) F (5-day) S (7-day) F (7 day)
12 12 12 29
65 63 5 3 83 58 85 2 2 99 70 00 f 2 04 70 03 5 3 20
61 85 k 6 13 57 53 4 39 62 01 285 64 70 -t 4 15
1 8 7 5 013 2 13 2 0 18 1645012 1705013
mg
5
kgicm
6 52
t 5 13*
t 4 49
*
'
' P < 0 05 **P < 0 01
72
Muscle Properties after Weightlessness
MUSCLE . 8 NERVE
January 1991
S
F
I
A
1
SOLEUS
cross-sectional area and expressed in kg/cm2. The tensions per surface unit were not significantly changed either in the slow soleus muscle or in the fast gastrocnemius and plantaris muscles. This meant that atrophy was the main cause of the force decrease.
T h e relationships between the relative tension (P/P,) and the calcium Concentration expressed in pCa values were established for the soleus, the gastrocnemius and the plantaris. They were called T/pCa relationships with -I‘defined as the P/P,, ratio. T h e results obtained for the 1667 spaceflight are illustrated in Figure 2. T h e curves were established from the Hill equation5: PIP,, = [Ca]” / (K” + [Ca]”) where n is the Hill coefficient and K is the apparent dissociation constant. From these curves, different parameters were determined. T h e Ca2+ threshold for activation of the contractile proteins was defined as the lowest Ca2+concentration required to obtain the development of a tension. Then, the position of the ‘T/pCa curves could be characterized by the pCa,, value which corresponded to the midpoint of the ‘l’/pCa curve, ie, the pCa value at which half-maximal tension was elicited. This parameter indicated the apparent Cay+ affinity. Moreover Hill plots (Fig. 3 ) were used to determine the steepness of the curves. From each Hill plot, two Hill coefficients were measured: n , for the slope of the curve when PIP,, was more than 0.50 and n2 for the slope when P/P, was less than 0.50. Finally, all the results are summarized in Table 3. For the soleus muscle, the activation threshold was higher after the 1667 spaceflight. It was respectively equal to pCa 6.40 and pCa 6.60 for flight (F) and synchronous (S) animals. This meant that after flight the fibers started to contract with a higher calcium concentration when compared to control. T h e pCa,,, values were decreased from 5.90 (S) to 5.75 (F). In Figure 3 it appeared that n I and n2 parameters were higher after the 1667 spaceflight (see Table 3 ) . T h e ‘T/pCa relationship established for the gastrocnemius showed a slightly higher threshold f o r calcium activation (Table 3 ) . ‘l’his threshold was equal to 6.30 (S) and 6.20 (F) after the 7-day spaceflight (Fig. 2U). As described for the soleus, the curve was shifted toward lower pCa values and the pCa.,[) values decreased from 5.97 (S) to 5.87 (F) in the 7-day flight. A shift by 0.15 pCa unit was measured after the 5-day flight. The n I values
TensionlpCa Relationships.
4.8
R
4.8
R
FIGURE 1. Records of maximal tensions induced by a pCa 4.8 solution on single-skinned fibers of soleus, gastrocnemius lateralis, and plantaris muscles for synchronous (S) and flight (F) animals (flight 1667). When the steady maximal tension was reached, the fiber was relaxed by the “R” solution. The fast transient peaks on the records and the transient displacements of the base line are produced by the chgnges of the solutions. The reference level used for the amplitude measurements is shown by a dashed line just before the record.
for the soleus muscle after spaceflights whereas they remained unchanged for the gastrocnemius and plantaris muscles. T h e results of both flights are summarized in Table 2. The soleus muscle fibers exhibited a clear decline in the absolute maximal tension expressed in mg (up to 28% after the 1667 biocosmos). T h e gastrocnemius showed a significant modification when the tension was expressed in milligrams after the 1514 spaceflight while a less important decrease appeared after the 1667 spaceflight (not significantly different from the controls). In the plantaris muscle, no change was observed after both spaceflights. All these results suggested that atrophy particularly occurred in the slow muscle. Since it is well known that tension is dependent on the fiber size, the maximal tensions were also calculated in relation to the
Muscle Properties after Weightlessness
MUSCLE & NERVE
January 1991
73
-
n 0
100-
50
Table 3. Parameters of the TlpCa relationships
A
.
Activation threshold (APW
F
4 W
ApCa,,
a
0
-
"1
B -
n2
0
a 100- B
z 0 u)
bI-
Sol GL PL Sol GL PL Sol GL PL Sol GL PL
ControllFlight 1514
ControllFlight 1667
-
0 20 0 10 0 0 15 0 10 0 1 6611 81 2 7712 88 2 7012 70 3 0013 14 5 8814 54 4 9014 90
0.10 0 -
0.15 0 -
2.6612.40 2.6612.66 -
5.7114.00
5.0015.00
S o l , G L , and P L designed respectively the soleus. the gastrocnemius lateralis, and the plantans muscles Results for the activation threshold and the pCa,, parameters were expressed in terms of differences between control (synchronous) and flight values 0 indicated no difference Dash indicates no result n, and np were the slopes of the fitted line above and below 50% of PiPo respectively
W
2 -I
W
II:
6.4
5.6
4.8
change occurred in the tension/pCa relationship. T h e n , and n2 parameters remained unchanged.
z
8z
50.
W
I-
PLANTARIS
W
2
4 w
II:
FIGURE 2. Relative tensionlpca relationships in soleus (A), gastrocnemius lateralis (B), and plantaris (C) muscles for synchronous (filled circles) and flight (open circles) fibers. Curves were drawn through the experimental points according to the Hill equation using n, and n2 Hill parameters (see text). Values are means 2 SEM. The number of fibers in each curve was the same as listed in Table 2 for the 7-day results.
were only slightly modified -respectively decreased o r increased after the 15 14 and the 1667 spaceflights ('Table 3). This slight modification indicated that this part of the relationship (>pCa,,,) was not clearly changed by the flights. On the contrary, the n2 values were clearly decreased for both flights. T h u s the influence of the spaceflights seemed more apparent when the fi-ee Ca2+ concentration was relatively low. T h e plantaris muscle was unaffected by the 2 spaceflights. Similar thresholds equal to 6.30, similar pCa,,, values equal to 5.85 were measured both in the synchronous and flight animals. N o
74
Muscle Properties after Weightlessness
An attempt to compare the tension kinetics in synchronous and flight animals was made by measuring the time to achieve the maximum tension level (t,,,',,). We were aware that this parameter might be an imperfect indicator of muscle fiber velocity in skinned fiber. However, o u r purpose was only to determine the possible effect of the flights essentially, from a qualitative point of view. l h e data from the three muscles for the 1667 flight (7 days) are summarized in Figure 4. Similar results were obtained after the 1514 flight (5 days). T h e rate of force development observed on the synchronous animals for the three muscles depended on the amplitude of the tensions elicited by the different pCa solutions. T h e higher the pCa value, the lower the tension and the larger the t,,,,,. In synchronous animals, the values of t,,,',, depended on the muscle. For the maximal tensions, t,,,,, varied as follows: plantaris ( 3 sec) < gastrocnemius (6 sec) < soleus (12 sec). These values are in good agreement with those generally proposed in ratskinned fibers.20 In the soleus muscle, the tensions developed faster after spaceflight (Fig. 4A), which was true whatever the initial tension amplitude. In the gastrocnemius, the spaceflight induced a slight decrease in the rate of force development (Fig. 4B). In the plantaris (Fig. 4C) the rate of contraction Rate of Force Development.
MUSCLE & NERVE
January 1991
firmed that a 5 or a 7-day spaceflight was long enough to induce changes in the contractile proteins.
A consistent atrophy appeared in the soleus muscle since we found a significant decline in muscle mass and fiber diameter. A significant decrease in the gastrocnemius and plantaris muscle mass also happened after both flights while in the gastrocnemius the decrease in fiber diameter clearly occurred only after the 1514 flight. So, as generally reported,'"."'3" the atrophy was more pronounced in the slow soleus muscle.
Muscle Atrophy.
-3'. . 7.2
-
*
6.4
.
-
-
5.6
-
.
'
4.8
B
7
s
a -3l
.
7.2
.
1
6.4
5.6
4.8
C n
l i
-* 7.2
6.4
5.6
0 6.4
5.6
4.8
0' 6.4
5.6
4.8
5.6
4.8
4.8
pCa FIGURE 3. Hill plots of the mean values of the pCa-force data for fibers from the soleus (A), the gastrocnemius lateralis (B), and the plantaris (C) muscles (number of fibers as in Fig. 2 in each group). Synchronous fibers (filled circles) and flight fibers (open circles).
I
always decreased significantly after the spaceflights. DISCUSSION
Spaceflights have been well known to induce muscle a t r o p ~ y ~ ~ . 7 . 1 : ~ , ~ 0and . 2 1 most of the results were generally obtaiqed after 18- to 20-day spaceflights. A major point emphasized in this article is that space atrophy and physiological changes happened after spaceflights as short as 5 or 7 days. Recently, biochemical changes after a 7-day flight (SL3) have been described." Moreover on the hindlimb suspended rat, the half-time of the degradation rate constant in the soleus was 4.1 days for the myofibril proteins and 3.6 days for the slow myosin content.") Therefore, our results con-
Muscle Properties after Weightlessness
0' 6.4
PCa FIGURE 4. Relationships between tmaX the time to reach the maximum level of the tension and pCa in soleus (A), gastrocnemius lateralis (B), and plantaris (C) muscles for synchronous (filled circles) and flight (open circles) fibers. Values are means +. SEM. Number of fibers as in Figure 2.
MUSCLE & NERVE
January 1991
75
The changes in the fast muscles were less marked. However we must point out that the flights had more marked effects on the gastrocnemius, a fast muscle with an antigravitational function than on the plantaris, a fast-twitch muscle used in ankle movement. T h e decline in fiber size has usually been described in various disuse atrophy situations such as immobilization (casting, bed rest, or water immersion), denervation, and tenotomy. It was generally explained by a decrease in the content of myofibril proteins. The main explanation might be a reduction in the rate of protein synthesis in conjunction with an elevation in the rate of protein degradation." When the muscle loss was not associated with a fiber diameter decline, it was attributed primarily to a loss of muscle tissue Our results showed changes in the maximal tension dependent on muscle type. A very significant decrease in P,, expressed in milligrams appeared in the soleus as already described on glycerinated fibers."',21 T h e decrease was more variable for the gastrocnemius (significant for the 1514 flight and not significant for the 1667 one) and the fiber type heterogeneity of this muscle might be responsible for the difference between the two flights. N o modification occurred in the plantaris, a fast muscle. This was already described for the EDI, (extensor digitorum longus), another fast muscle after biocosmos 936 (18.5 These authors even reported an increase in EDL tension after another biocosmos (1 129 flight, 18.5 days). Moreover the decrease in absolute values of P,, disappeared when the force was expressed per cross-sectional area. Since a decrease of maximal tension was usually related to a decrease of the maximal cross-bridge number between actiri and myosin, our result indicated that a decrease in the number of cross-bridges was more probable than a change in density or a change of force per cross-bridge. S o , the decrease of muscle strength after a spaceflight could result from muscle atrophy. T h e data from the TlpCa curves indicated that the value of the Ca2+ threshold for activation in the soleus and in the gastrocnemius was higher after spaceflights. So, the sensitivity of' the myofilaments for Ca2+ was decreased. T h e apparent Ca2' threshold for activation was well known to be higher for the fast fibers,11,2(i the highest threshold being obtained for intermediate fibers." 'l'herefore, our result suggested that the slow soleus muscle appeared to
Ca2+ Affinity of the Contractile Proteins.
76
Muscle Properties after Weightlessness
get Cay+ threshold activation characteristics that resembled more those of a fast muscle. On the other hand, the initially fast gastrocnemius lateralis muscle might modify its characteristics towards those of an intermediate muscle. No significant change in the calcium threshold for the plantaris was observed. Moreover, we described a shift of the tension/ pCa relationships of the soleus and gastrocnemius toward lower pCa values while that of the plantaris remained unchanged. T h e position of the T/ pCa curve was generally assumed to be directed by the Ca2+ binding properties. Indeed, an increase in the isometric tension in skinned fibers was presumed to parallel the binding of Ca'+ to regulatory sites on troponin C.I6 Thus, the shifts toward the right of the T/pCa curves for soleus and gastrocnemius after flights were assumed to parallel the decreases in the apparent Ca2+ affinity of the tropinin C, illustrated by the decrease of pCa,,, values. Another result was the Hill coefficient measurements, n , and n2, representative of the steepness of the T/pCa relationships. Two coefficients were necessary to obtain the best fit between the theoretical curve and our experimental values: n , for the curve above pCa,,, and n2 for the curve below pCa,,,. A similar analysis was already used by different authors. When changes occurred, they were more obvious for the n2 coefficient. In most current models of muscle activation, the value of the Hill coefficients reflected the cooperativity among Ca2+ binding sites along the thin filament. So, the increase observed in the soleus, especially for the n2 value might be explained by a greater cooperativity. Then, the soleus muscle seemed to react more like a fast muscle. In the gastrocnemius the changes of the n , values were not homogeneous (slight decrease after the 1514 flight and slight increase after the 1667 flight) while the n2 values were clearly decreased after both flights. This should indicate a lower cooperativity of the Ca2+ binding sites in the gastrocnemius muscle more characteristic of a slower muscle. In plantaris, n , and n2 values remained unchanged after both flights. Experiments of partial substitution with troponin C from different types".'7,''' clearly showed that the position and steepness of the T/pCa curves were strongly dependent on the type of troponin C present in the muscle. This indicated that troponin C played a direct role in the cooperative mechanism.5 However, our values of n,, especially in the fast muscles as well as the nE3values
MUSCLE & NERVE
January 1991
previously obtained by Brandt et a14 on the rabbit psoas ( n Hfrom 5.0 to 5.6) were too large to be explained by a control mechanism governed exclusively by
