INTRODUCTION

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A MAJOR PROBLEM ASSOCIATED WITH space travel is the multifaceted deterioration of limb skeletal muscle (5, 26, 31). Skeletal muscles from rats flown in space have shown fiber atrophy, degeneration of motor innervation, muscle fiber segmental necrosis and central-core lesions, and disruption of the microvasculature, with the greatest change observed in antigravity muscles such as the soleus (Sol) (18, 26). For example, the relative atrophy observed with both models of weightlessness and 0 gravity (G; magnitude of the force of gravity on the surface of the Earth) was Sol > gastrocnemius = plantaris > extensor digitorum longus (8, 18, 28). Considerably less is known about the effects of weightlessness on skeletal muscle function in humans and nonhuman primates. During Skylab flights, the crew experienced a 12% decrease in leg volume and a 20% decrease in muscle strength (5). The greater decline in strength relative to muscle size suggests that factors in addition to cell atrophy contributed to the strength loss. Models of weightlessness employing rodents have clearly demonstrated that both peak force (N) and tension [N/fiber cross-sectional area (CSA)] decrease in response to unloading (11, 19-21). The reduced tension is thought to be caused by a selective loss in contractile proteins, such that the number of active cross bridges per CSA declines (29).

To assess the cellular basis of the functional changes induced by weightlessness, we have used the single skinned-fiber preparation and the hindlimb unloading (HU) rat model (11, 19-21). In addition to fiber atrophy and the decline in force, HU has been shown to increase maximal fiber shorterning velocity (V_o) and ATPase and reduce peak power in the slow type I fibers of the Sol (19, 20). The percentage of fibers expressing fast myosin heavy chain (MHC) increased from 4 to 29% by 3 wk of HU; however, the majority of the fibers showed a significant increase in V_o while maintaining an exclusively slow type myosin isozyme profile (20).

Clearly, there is a need for additional information about the cellular adaptations induced by 0 G in humans and/or other large nonhuman primates before scientifically sound exercise countermeasures can be developed. Without the latter, it will be impossible to safely conduct the prolonged space missions required for exploration of Mars and other points of the universe. Recently, US and Russian scientists have studied the effects of spaceflight on the neuromuscular system in rhesus monkeys (2). The general hypothesis is that the rhesus monkey may be an excellent model for studying not only the mechanisms of 0-G-induced muscle atrophy but also the adaptive responses and strategies of the neuromuscular system. To date there is a scarcity of information concerning the functional properties of limb skeletal muscles in rhesus monkeys. Before alterations induced by 0 G can be studied and understood, it is important to understand the monkey's normal physiology. Consequently, the purpose of this work was to characterize the fiber type distribution and size characteristics of the slow Sol and fast gastrocnemius muscles of adult rhesus monkeys and determine the force, velocity, power, and Ca²⁺ sensitivity of individual fast- and slow-twitch fibers. A second purpose was to determine the effect of the flight facility itself on contractile function. Because the animals must fly in an experiment-support primate facility [Experimental System for the Orbiting Primate (ESOP)], it is important to know whether the reduced mobility imposed by the ESOP had any direct effects on muscle function.

	METHODS

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Selection of animals and general care. The study described herein was conducted as part of a large joint effort between the US and French space agencies [National Aeronautics and Space Administration (NASA) and Centre d'Etudes Spatiales, respectively] designed to develop baseline ground-based data on the basic physiology and psychomotor performance of adult rhesus monkeys (Macaca mulatta). The project was approved by the animal care and use committee at NASA-Ames Research Center (Moffet Field, CA) and Marquette University (Milwaukee, WI).

Biopsy procedure. Muscle biopsies were obtained 4 wk before (pre) and immediately after (post) the 18-day ESOP sit. The pre- and postbiopsies were taken from two independent sites in the Sol and the MG muscles by using an open-biopsy technique. All biopsies were taken from the right leg, and the specific location of each biopsy site is shown in Fig. 1. The sites were selected to ensure that the same muscle fibers were not sampled during the pre- and postflight biopsies, as determined from detailed architectural analyses of each muscle (27). Additionally, the regions sampled within each muscle have been shown to have similar fiber type distributions (27).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Schematic drawing of lower limb (medial view) of Macaca mulatta illustrating relative locations of pre ()- and post ()-experiment support primate facility [Experimental System for the Orbiting Primate (ESOP)] biopsies in soleus (Sol) and medial gastrocnemius (MG). Muscle fibers of Sol extend down to calcaneus, and both muscles can be isolated on medial side of limb (modified from Ref. 2).

Histochemical identification of fiber type. Fibers were classified as type I, IIa, IIx, or type I/II (hybrid; coexpression of slow and fast myosin) by using monoclonal antibodies that label the different MHC isoforms. Serial cross sections were incubated with primary antibodies [BA-F8, BF-13, BF-35, and SC-71 generously donated by S. Schiaffino (Padua, Italy)] overnight at 25°C. Sections incubated without primary antibody were used as a control to visualize nonspecific labeling. A Vectastain ABCt kit (Vector Labs, Burlingame, CA) was used to amplify the antigen-antibody complex, which, in turn, was visualized by treatment with a diaminobenzidene peroxidase reaction.

Single-fiber preparation. On the day of an experiment, a muscle bundle (Sol or MG) was transferred to a dissecting chamber containing relaxing solution pCa 9.0 (4°C, where pCa = log Ca²⁺ concentration) that contained the following (in mM): 20 imidazole, 7 EGTA, 10.0 caffeine, 14.5 creatine phosphate, 4.74 ATP, 5.40 MgCl₂, and 0.016 CaCl₂ · 2H₂O. A single fiber was isolated from the bundle and transferred to an experimental chamber containing relaxing solution. This stainless steel chamber contained three troughs that allowed the fiber to be moved from a low-Ca²⁺ relaxing solution to activating solutions of various pCa values. A fiber segment (~2 mm long) was mounted between a force transducer (model 400, Cambridge Technology, Cambridge, MA; sensitivity 2 mV/mg) and an isotonic direct-current torque motor (model 300H, Cambridge Technology) as described previously in detail (32). The fiber was briefly bathed in relaxing solution containing 0.5% (wt/vol) Brij-58 (polyoxyethylene 20 cetyl ether, Sigma Chemical, St. Louis, MO) before study to inhibit any residual sarcoplasmic reticulum Ca²⁺ uptake activity. The experimental chamber was mounted on the stage of an inverted microscope, and temperature was maintained at 15°C. Sarcomere length was adjusted to 2.5 µm by using an eyepiece micrometer (×800), and segment length was determined by moving the microscope stage with a micrometer so that the fiber segment moved across the visual field of the eyepiece. The segment length was determined directly from the micrometer displacement. A Polaroid photograph was taken of the fiber while it was briefly suspended in air. Fiber width was determined at three points along the length of the photograph, and fiber CSA was calculated from the mean width, assuming the fiber forms a circular cross section when suspended in air. Any fiber showing a high degree of striation nonuniformity or a damaged region was discarded.

Determination of peak force and stiffness. The outputs of the force transducer and position motor were observed on a digital-storage oscilloscope and then amplified and interfaced to a microcomputer (Lab Master DMA input-output board, Scientific Solutions, Solon, OH). Custom-designed software performed online analysis as described below. Force in relaxing solution was monitored, and the fiber was activated by transfer into activating solution (pCa 4.5) that contained the following (in mM): 20.0 imidazole, 7.0 EGTA, 10.0 caffeine, 14.5 creatine phosphate, 4.81 ATP, 5.26 MgCl₂, and 7.0 CaCl₂ · 2H₂O. Both relaxing and activating solutions contained sufficient KOH and KCl to bring pH to 7.0 and total ionic strength to 180 mmol/l. The composition of the relaxing and activating solutions was determined by using the computer program of Fabiato and Fabiato (7) that uses the apparent stability constants reported by Godt and Lindley (13). Peak force (N) was determined in each fiber by computer subtraction of baseline force from peak force attained during maximal Ca²⁺ activation, and force (kN/m²) was calculated. Peak fiber stiffness was measured by applying small-amplitude sinusoidal changes in fiber length (FL; L) at a frequency of 1.5 kHz and measuring the magnitude of the resultant change in force (P). The elastic modulus (E_o) of each fiber was calculated as follows: E_o = P/L × FL/fiber CSA. Peak stiffness was determined by subtraction of the stiffness recorded in relaxing solution from that measured during full activation (pCa 4.5), exactly as described previously (21).

Determination of fiber V_o. V_o was determined by the slack test, as previously described in detail (32). Briefly, the fiber was activated, allowed to attain peak force, and then rapidly shortened by a predetermined length step so that force dropped to zero. There was a rapid redevelopment of force once the fiber had shortened to take up the imposed slack. The fiber was then returned to relaxing solution and reextended to its original FL. This entire procedure was repeated so that each fiber was subjected to a minimum of five different slack distances, each 20% of FL. The computer plotted the slack step distances against the corresponding times required for the redevelopment of force and fit the data with a first-order least-squares regression line. The slope of this line was V_o (in mm/s), which was normalized to FL at a sarcomere spacing of 2.5 µm (FL/s).

Force-velocity-power relationships. In a subset of fibers, the segment was subjected to a series of isotonic contractions as follows. The mounted fiber was transferred into a chamber filled with activating solution maintained at 15°C and allowed to attain peak isometric force (P_o). The position motor then rapidly stepped the fiber to three submaximal isotonic loads. The rate of fiber shortening was controlled by a servo- mechanism similar to that described by Julian and Moss (17). The duration of each isotonic step varied from 80 to 120 ms and was adjusted so that the total shortening that occurred over all three steps did not exceed 20% of FL. After the third isotonic step, the fiber was returned to relaxing solution and reextended to its original FL. The entire procedure was repeated until each fiber had been subjected to a total of 15-18 different isotonic loads.

Force-pCa relationships. Force-pCa relationships were determined by measuring P_o attained in activating solutions having free Ca²⁺ concentrations ranging from pCa 6.8 to 5.0. These solutions were made by mixing appropriate volumes of the maximal activating (pCa 4.5) and relaxing (pCa 9.0) solutions described above. The submaximal forces attained during these contractions, P_r, were expressed as a fraction of the peak isometric force determined in pCa 4.5, i.e., P_r = (submaximal P_o)/(P_o at pCa 4.5). Every fourth or fifth contraction was performed at pCa 4.5. Separate Hill plots [where log P_r/(1  P_r) is plotted against pCa] were fitted to data points <0.5 P_r and >0.5 P_r. The free-Ca²⁺ concentration that elicited half-maximal activation was defined as the mean abscissal intercept of each plot. The Ca²⁺ activation threshold was defined as the free-Ca²⁺ concentration obtained when the plot of points <0.5 P_r was extrapolated to a value of 2.5.

MHC composition. After the contractile measurements, the MHC composition of each fiber was determined by SDS-PAGE. The segment was removed from the experimental apparatus and solubilized in 10 µl of 1% SDS sample buffer containing 6 mg/ml EDTA, 0.06 M Tris, 1% SDS, 2 mg/ml bromophenol blue, 15% glycerol, and 5% -mercaptoethanol. The sample buffer (2.5 µl) was loaded on a Hoefer SE 600 gel system that consisted of a 3% (wt/vol) acrylamide stacking gel and a 5% (wt/vol) separating gel (25). Gels were silver stained as described by Giulian et al. (12). The relative content of each MHC in hybrid fibers was determined by densitometric scanning (CliniScan 2, Helena Laboratories, Beaumont, TX).

Statistical analysis. Data are presented as means ± SE. A t-test was used to determine intergroup differences in contractile properties. Statistical significance was accepted at P  0.05.

	RESULTS

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Fiber diameter and percent distribution. The fiber type distribution was determined by immunohistochemical staining to monoclonal antibodies as described in METHODS. A representative micrograph is shown in Fig. 2 and the percent fiber type distribution in Table 1. Consistent with other species, the monkey Sol contained primarily the slow type I fiber; however, the fast fiber population (IIa and hybrid) showed a higher percentage than we had previously observed in rats and humans (9, 10, 20). The MG contained primarily fast fibers, with the greatest percentage being type IIx. Interestingly, the monkey Sol and MG, similar to human muscle, contained no fast type IIb fibers. The slow type I fibers of the MG were significantly smaller than the Sol type I and Sol and MG type II fibers. This difference was apparent when fiber size was determined from a CSA analysis of the histochemically stained sections (Fig. 3) or from the diameter of the individual skinned fibers (Table 2). The latter technique is more sensitive and reliable because the diameter is measured at a constant sarcomere length of 2.5 µm. The ESOP sit caused a significant reduction in the diameter of the type I and hybrid fibers of the Sol and the type II and hybrid fibers of the MG, whereas the Sol type II and MG type I fibers were unaffected (Table 2; see Table 4; Fig. 3). The ESOL sit caused no major shifts in the fiber type distribution; however, we did observe an increased number of hybrid fibers postsit (Table 1; see Table 4).

View larger version (110K): [in this window] [in a new window]   Fig. 2.   Representative micrograph showing monoclonal antibody reactions for Sol and MG cross sections (see text for details).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Mean fiber cross-sectional area for ( A) soleus and (B) MG pre- and post- 18-day ESOP occupancy by aniamls in sitting position (ESOP sit). Solid, open, horizontally striped, and cross-hatched bars: type I, IIa, IIx, and I/II (hybrid) fibers, respectively; error bars, SE. * P < 0.05 vs. corresponding pre-ESOP mean.

Fiber force (N and kN/m²) and stiffness. Peak force (mN and kN/m²) for each fiber type is shown in Table 2. There were no major differences between the fiber types in their ability to generate force (kN/m²), although the Sol slow type I and fast type II fibers showed the lowest and highest force, respectively. The ESOP sit significantly reduced the force of the Sol type I and MG type II fiber types. This decline was entirely explained by the significant atrophy of these fiber types because the force per CSA (kN/m²) was not altered by the ESOP sit. Fiber stiffness averaged 2.39 ± 0.24 and 2.07 ± 0.37 kN/m² × 10⁴ in the slow fibers and 0.87 ± 0.11 and 1.09 ± 0.23 kN/m² × 10⁴ in the fast fibers of the Sol and MG, respectively. The ESOP sit had no effect on fiber stiffness.

V_o. A representative polyacrylamide gel illustrating MHC isoform expression in isolated single monkey fibers is shown in Fig. 4. The legend of Fig. 4 indicates the muscle source, and V_o is given below each lane. Lane 1 represents a hybrid fiber containing both slow and fast MHC and a V_o intermediate between the slow (lanes 2, 3, and 5) and fast (lane 4) fibers. Unlike with fibers in rats and humans, the various fast fiber types did not separate but rather migrated as a single band on the gel (Fig. 4). For Sol, this did not present a problem because the immunohistochemical results demonstrated that the type II fiber population to be entirely type IIa (Table 1). Consequently, the data for Sol type II fibers shown in Table 2 can be considered type IIa. The mean V_o of 4.19 ± 0.39 FL/s was significantly higher than that of the slow type I fiber from either Sol or MG but was lower than that of the MG type II population (Table 2). However, this latter group contained both IIa and IIx fibers, and, at least in humans, the IIx fiber is known to have higher V_o values than does the IIa fiber. If one assumes that the MG type II fibers with V_o values >6.0 (see Fig. 6) were type IIx fibers, then the type IIa and IIx fibers showed a mean V_o of 3.26 and 7.70 FL/s, respectively.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Representative 5% polyacrylamide gel demonstrating myosin heavy chain (MHC) identification in single rhesus Sol fibers. Each lane represents a single fiber, with fiber maximal shortening velocity (Vo) in fiber lengths (FL)/s shown below each lane. Lane 1, hybrid fiber containing both slow type I and fast type II MHC; lanes 2, 3, and 5, slow type I; lane 4, fast type II fibers.

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View larger version (20K): [in this window] [in a new window]   Fig. 5.   Frequency distributions for Vo of Sol fibers studied pre- (A) and (B) post-ESOP sit.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Frequency distributions for Vo of MG fibers studied pre- (A) and (B) post-ESOP sit.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Plot of fiber Vo vs. MHC type II-to-total MHC ratio. Closed and open symbols, individual fibers isolated from pre- and post-ESOP sit biopsies, respectively; large open squares, mean values for type I and type II fibers (fibers from Sol and MG, pre- and post-ESOP sit); vertical lines above and below large open squares, range for each fiber type; solid line, best fit to individual data points. (See Eq. 1 in text).

Isotonic contractile properties of rhesus monkey muscle fibers. The force-velocity relationship of a representative type I fiber, obtained from the Sol of an adult rhesus monkey maintained under normal living conditions, is presented in Fig. 8A. Three parameters are necessary to describe this relationship: 1) the intercept of the curve with the force axis, or P_o; 2) the velocity axis intercept (V_max); and 3) the dimensionless parameter a/P_o, which describes the shape or curvature of the relationship. In this particular example, P_o, V_max, and a/P_o values were 0.77 mN, 0.70 FL/s, and 0.029, respectively. Overlaid on the force-velocity curve is the force-absolute power relationship of the fiber. Peak power is attained at a unique point along the force-velocity relationship where the product of force (P) and shortening velocity (V) is maximal. In the present example, these conditions were attained at 14% of P_o, or 0.112 mN. At this external load, the fiber shortened at a velocity of 0.100 FL/s, producing a peak power output of 11.2 µN · FL · s¹.

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Force-velocity and force-power relationships of representative muscle fibers obtained from adult rhesus monkeys. A: results for 2 single Sol type I fibers, 1 obtained during control living conditions (solid line) and 1 obtained post-ESOP sit (dotted line). B: results for a single type II Sol fiber obtained during control living conditions. Note that velocity axes of A and B are scaled identically, whereas power axes vary by a factor of 5. See RESULTS for further details.

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Effect of ESOP occupancy on isotonic contractile properties. Force-velocity and force-power parameters of single fibers obtained immediately after 18 days of ESOP occupancy are compiled in Tables 3 and 4. The force-velocity parameters (V_max and a/P_o) were not affected by the ESOP sit in either muscle or fiber type. Consequently, the relative submaximal force and velocity that elicited peak power and the absolute shortening velocity at peak power output were also unchanged. However, the absolute force associated with peak power declined in parallel to the loss of fiber P_o, i.e., by 19% for Sol type I fibers and by 26% for MG type II fibers (Table 3). These alterations in force-velocity parameters are illustrated by post-ESOP Sol type I fibers plotted in Fig. 8A. Although V_max (0.77 FL/s) and a/P_o (0.026) of this post-ESOP fiber were similar to the values noted for the control fiber, its P_o was 23% lower (0.59 mN) and its peak power was reduced by 25% (8.4 µN · FL · s¹). Overall, peak power was reduced by an average of 25 and 18% for Sol type I and MG type II fibers, respectively (Table 3). Because of the greater variability in the average type II fiber peak power, only the change for Sol fibers was statistically significant.

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View larger version (21K): [in this window] [in a new window]   Fig. 9.   Peak absolute power-frequency distributions of Sol and MG fibers from adult rhesus monkeys. A: Sol fibers obtained during control conditions. B: Sol fibers obtained post-ESOP sit. C: MG fibers obtained during control conditions. D: MG fibers obtained post-ESOP sit.

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Fiber Ca²⁺ sensitivity before and after ESOP occupancy. Force-pCa relationships for Sol type I and MG type I and II fibers (there was an insufficient number of Sol type II fibers and hybrid fibers for analysis) are presented in Fig. 10. For all groups, relative force rose rapidly at free-Ca²⁺ concentrations between pCa 6.5 and 5.5. However, the force-pCa relationship for the type II fibers was shifted to the right, and relative force appeared to rise more rapidly with increments in free Ca²⁺.

View larger version (13K): [in this window] [in a new window]   Fig. 10.   Force-pCa relationships of single fibers obtained from adult rhesus monkeys. A: Sol fibers expressing type I MHC. B: MG fibers expressing type I MHC. C: MG fibers expressing type II MHC.  and solid lines, Fibers obtained during normal ambulatory living conditions;  and dotted lines, fibers obtained post-ESOP sit; error bars, ±1 SE. P, force; Po, peak isometric force.

	DISCUSSION

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A major challenge facing the field of space biology is to identify the cellular and molecular alterations in limb skeletal muscle with weightlessness and ultimately to develop effective countermeasures. Rhesus monkeys offer a number of advantages over other smaller animals such as rats in that comprehensive studies of sensory-motor control and cellular responses to 0 G are possible. However, to date there are very few data concerning the functional properties of monkey limb skeletal muscle (6). The results of this study address this void and provide important information on how the flight-chair environment in itself alters cell function.

Fiber type distribution, size, and peak force. In agreement with previous observations in monkeys (1, 2, 6, 27) and other species (10, 24), we found that Sol and MG muscles contain primarily slow type I and fast type II fibers, respectively. Although the rhesus monkey Sol has been reported to be 90% slow-twitch type I (27), three studies including ours have found the percentage of slow type I fibers to range from values in the low 60s to the high 70s (2, 6). Thus it appears that rhesus monkey Sol contains a higher percentage of fast fibers (all of which are fast type IIa fibers) than generally observed in rodents or humans (9, 10, 24). Nevertheless, we have recently shown that the fiber size and contractile properties of the type I fibers from rhesus monkey Sol more closely resemble those in humans than they do of those in rat Sol (33). When results from humans, rhesus monkeys, and rats were compared, the fiber force per CSA was found to be independent of species size and relatively constant across species (9, 33). In contrast, V_o and peak power were inversely related to species body mass (33). These data support the hypothesis that the rhesus monkey would be a good model for assessing the effects of weightlessness on human muscle function. Consistent with a previous report (2), we observed by two independent methods that MG type I fibers were significantly smaller than was any other fiber type. This difference has also been observed in humans but not in rats (9, 33). However, the extent of the effect was greater in monkeys, such that the mean diameter of the MG type I fiber observed herein was smaller than of a similar fiber population in rats (33). This is an exception to the general observation of a small increase in fiber diameter with species size (33). The small fiber size in MG type I fibers caused the peak force to be low compared with that of the other fibers studied (Table 2). However, the force per CSA was similar to that of the other fiber types, which suggests that the myofilament content per CSA was identical between fiber types. Further support for this comes from the observation that fiber stiffness in Sol and MG type I fibers was similar. Because fiber stiffness, at least in part, reflects the number of attached cross bridges, it is apparent that no major difference in cross-bridge content existed between these fiber populations. It is not clear why monkey MG type I fiber diameter fell below that expected for the species size, but one possibility relates to the normal weight-bearing activity of this species. Monkeys spend a considerable amount of time in a squatting posture. Consequently, the triceps surae is placed in a shortened position, with the biarticulate MG likely to be more affected than the Sol. Placing the muscle in a chronically shortened position would be expected to reduce reflex activation of the muscle, and the resulting inactivity could induce fiber atrophy relative to the type I population of the Sol. The fact that the muscle was still weight bearing likely protected against a loss of contractile protein, and thus peak force (kN/m²) was maintained.

V_o. V_o for the monkey slow type I and fast type II fibers were intermediate compared with that in fiber types in rats and humans. For example, the monkey Sol type I fiber V_o reported here (Table 2) was between the 1.34 and 0.52 FL/s published previously for rats and humans, respectively (33). MG type II fibers were faster than Sol type II fibers, undoubtedly due to the latter being entirely type IIa whereas the former contained a mixture of IIa and IIx fibers. We know from data in humans that the type IIx fiber contains a significantly higher V_o than the type IIa fiber (34). The hybrid fibers showed a V_o higher than that for type I but considerable lower than that for type II fibers. Fibers with a relatively high type II-to-total MHC ratio (0.6-0.8) had velocities only slightly higher than pure type I fibers. This observation is consistent with the hypothesis that in hybrid fibers the slow MHC would be expected to have a disproportionately greater influence on V_o (25). Because the myosin heads composed of slow myosin would detach considerably more slowly than fast myosin heads, they would provide a significant internal drag to fiber shortening speed. Cordonnier et al. (6) suggested that the V_o of hybrid fibers would be determined by the dominant species; thus fibers with a predominant fast MHC content would resemble pure fast fibers. Our results (Fig. 7) and those of Reiser et al. (25) suggest that this is not the case, that, in fact, even a small amount of type I MHC significantly reduces fiber V_o compared with that of a pure fast fiber (Fig. 7).

Force-velocity relationship and peak power. The greater peak power of the control fast fibers was due to the following characteristics of their force-velocity relationships: 1) type II fibers displayed a lower degree of curvature so that peak power was attained at a greater percentage of both V_max and P_o; 2) V_max was greater for the type II fibers; and 3) in the case of the MG only, type II fibers were capable of producing greater P_o (mN). These differences between the force-velocity parameters of type I and type II fibers in monkeys are qualitatively similar to those observed for single slow and fast fibers from other species, such as rats and humans (3, 35).

PCa-force relationship. In addition to differences in force-velocity parameters, type II fibers had a lower Ca²⁺ sensitivity than did type I fibers. This finding is in agreement with what others have reported for the rhesus monkey (6) and other species (14, 34). High-velocity fibers express fast isoforms of the regulatory protein troponin C (14). This isoform has two Ca²⁺-binding sites, vs. a single binding site on the slow troponin C isoform, and this may be responsible in part for the reduced Ca²⁺ sensitivity of fast fibers (23). It has also been proposed that the faster cross-bridge cycling rates of high-velocity fibers produce less cooperative activation of the thin filament compared with the slower cycling cross bridges of slow-velocity fibers (4). This would also tend to reduce the Ca²⁺ sensitivity of fast fibers.

Effects of the ESOP sit. The primary effect of the ESOP sit was to produce a small but significant fiber atrophy. Sol type I and MG type II fibers showed an ~10% atrophy, whereas the diameter of the hybrid fibers of Sol and MG declined by 15 and 28%, respectively. The immunohistochemical results (Fig. 3) suggest that MG type II fiber atrophy was confined to type IIa fibers. The apparent increase in the number of hybrid fibers is difficult to interpret because the immunohistochemical results showed an increased hybrid number in both the control and experimental group (Table 1). The most logical explanation is that the ESOP sit induced a small number of type I fibers in both Sol and MG to express type II as well as type I myosin. The small size of hybrid fibers (Table 4) and their velocity distribution (Figs. 5 and 6) suggest that the hybrids developed from type I rather than type II fibers. The increase in hybrid fibers or the expression of fast myosin in slow fibers is consistent with what has been observed in rats after periods of HU (20). Presumably, the reduced activity and/or loading of the muscle triggers the fast-myosin expression.

Summary. The peak force-generating capacity between the slow- and fast-twitch fibers in rhesus monkeys was not significantly different, nor was it different from values reported for other species. In contrast, V_o, V_max, peak power, and the pCa-force relationship were dependent on fiber type, and the values were closer to those in humans than to those in rats. This observation is consistent with the known inverse relationships of V_o, V_max, and peak power to body size. The major effect of the 18-day ESOP sit was to induce a small but significant decline in fiber diameter that, in turn, reduced the absolute magnitude of both peak force (mN) and power (µN · FL · s¹). This suggests that future 0-G studies evaluating the effects of weightlessness on rhesus monkey muscle function can be conducted with the confidence that any alterations in peak force and power per CSA or V_o are induced by 0 G and not the ESOP facility.