Effects of prolonged space flight on human skeletal muscle enzyme and substrate profiles

R. H. Fitts, P. A. Colloton, S. W. Trappe, D. L. Costill, J. L. W. Bain, D. A. Riley

Abstract

Our primary goal was to determine the effects of 6-mo flight on the International Space Station (ISS) on selected anaerobic and aerobic enzymes, and the content of glycogen and lipids in slow and fast fibers of the soleus and gastrocnemius. Following local anesthesia, biopsies were obtained from nine ISS crew members ∼45 days preflight and on landing day (R+0) postflight. We subdivided the crew into those who ran 200 min/wk or more (high treadmill, HT) in-flight from those who ran <100 min/wk (low treadmill, LT). In the LT group, there was a loss of lipid in soleus type I fibers, and muscle glycogen significantly increased in soleus fiber types postflight. Soleus cytochrome oxidase (CO) activity was significantly depressed postflight in the type I fiber. This was attributed to the LT group where CO activity was reduced 59%. Otherwise, there was no change in the crew mean for type I or IIa fiber glycolytic or mitochondrial enzyme activities pre- vs. postflight in either muscle. However, two of the three HT subjects (Subjects E and H) showed significant increases in both β-hydroxyacyl-CoA dehydrogenase and citrate synthase in the soleus type I fibers, and Subject E, exhibiting the largest increase in soleus oxidative enzymes, was the only subject to show a significant decrease in glycolytic enzyme activity. It is apparent that crew members performing adequate treadmill running can maintain calf muscle enzymes, which suggests that increased fatigue with weightlessness cannot be directly caused by a decline in muscle enzyme capacity.

  • enzymes
  • substrates
  • weightlessness
  • countermeasures
  • exercise

one of the major problems facing manned exploration of Earth's Moon, Mars, and destinations further out in the solar system is curbing muscle wasting, and the associated decline in performance, and increased fatigue that occurs with prolonged missions in microgravity. Since the Skylab flights in the 1970s and the MIR space station flights in the 1980s, it has been known that the microgravity environment leads to considerable muscle wasting and functional decline in limb muscles, with some of the most affected muscles being the soleus and gastrocnemius of the calf (11). Our recent studies of crew members after spending 6 mo on the International Space Station (ISS) showed that prolonged weightlessness produced substantial loss of fiber mass, force, and power, with the hierarchy of the effects being soleus type I > soleus type II > gastrocnemius type I > gastrocnemius type II (11). The quantitatively most important adaptation was fiber atrophy, which averaged 20% in soleus type I fibers but reached as high as 46% in one crew member. The loss of lower leg muscle circumference and performance observed during Skylab, MIR, and the more recent ISS flights occurred despite considerable countermeasure exercise (26).

The observation that three male crewmembers aboard the 84-day Skylab 4 mission were able not only to maintain but actually increase V̇o2peak, suggested that cardiovascular capacity could be maintained during prolonged microgravity missions (23, 25). However, during the 17-day STS-78 Shuttle mission, we observed a progressive decline of −6%, −9%, and −11% in the V̇o2 obtained while exercising at a heart rate (HR) equal to 85% of the preflight maximum HR on spaceflight days 2, 8, and 13, respectively (27). Furthermore, while Levine et al. (18) reported no change in V̇o2peak during 9- and 14-day space shuttle missions aboard SLS-1 and SLS-2, they reported a postflight average decline of 22%. Collectively, these data suggest that the earlier Skylab data may have been influenced by a training response due to the large amount of countermeasure exercise in-flight that far exceeded the crew's preflight exercise regimen (23, 25). The microgravity-induced decline in V̇o2peak appears to be, at least in part, caused by a decrease in plasma volume that reduces stroke volume and cardiac output (27). Regardless of the cause, the reduced V̇o2peak in microgravity would increase the relative workload of any task, and could in part explain the observation that astronauts performing heavy work such as extravehicular activity experience increased fatigability (18).

Work capacity and resistance to fatigue are dependent not only on aerobic capacity, but are also influenced by metabolic changes within the muscle. Microgravity causes a fiber type shift from slow type I to fast phenotypes, with the magnitude of the shift greater in prolonged vs. short-duration flights and correlated with the extent of fiber atrophy (8, 11, 28). For a given workload, the relative work for the atrophied fibers would be higher, which in turn would stimulate glycolysis and presumably trigger the induction of glycolytic enzymes. Data from rats flown in space provide evidence for a microgravity-induced increase in glycolytic enzymes. Manchester et al. (20) and Chi et al. (3) found selected glycolytic enzymes in the rat soleus to increase 28 to 56% following a 12.5-day spaceflight, while Musacchia et al. (24) reported lactate dehydrogenase (LDH) activity of the rat vastus medialis to increase 52% following a 14-day flight. Regarding muscle oxidative capacity, the data from rats show that the loss of mitochondrial protein with short-duration spaceflight is less than that observed for the contractile protein and for the degree of cell atrophy. Thus mitochondrial oxidative enzyme activity (per gram tissue dry weight) either remains unaltered or slightly elevated (10). Consistent with this, Edgerton et al. (8) found no significant effects of an 11-day spaceflight on the succinate dehydrogenase (SDH) activity of the vastus lateralis in humans. Despite the maintenance of muscle oxidative enzyme activity and the ability to oxidize pyruvate, Baldwin et al. (1) observed a 37% decline in the capacity to oxidize long-chain fatty acids in the high- and low-oxidative regions of the rat vastus muscle following a 9-day spaceflight. An increased reliance on glycolysis coupled with a reduced ability to oxidize fats would accelerate muscle glycogen use and increase metabolic products such as the H+ and inorganic phosphate (Pi) ions known to contribute to fatigue (6, 7, 16).

To date, there are no data on how prolonged spaceflight alters the metabolic or substrate profile in human skeletal muscle. The purpose of this work was to determine the effects of a 6-mo spaceflight on the ISS on selected anaerobic and aerobic enzymes, and the content of glycogen and lipids in slow and fast fibers of the soleus and gastrocnemius. The effects of countermeasure exercise were evaluated by relating pre- and postflight enzyme patterns to the extent of in-flight treadmill exercise.

METHODS

Flight and Subjects

The overall study design and subject characteristics were reported previously (11, 26) and are briefly reviewed here. The crew members participating in this study flew aboard the ISS from increments 5 to 11 (2002–2005). In the overall study group there were 10 crew members: 5 American astronauts and 5 Russian cosmonauts; however, for the metabolic studies described here there were nine subjects (5 astronauts and 4 cosmonauts). Due to small sample size and/or problems in shipment from Russia to the United States, the histochemical and biochemical assays were performed on biopsy tissue from six or seven and eight crew members, respectively. Prior to volunteering to participate in this study, all crew members were informed of the risks and benefits of the research and gave their written consent in accordance with the Human Subjects Institutional Review Boards at Marquette University, Ball State University, The Medical College of Wisconsin, and the National Aeronautics and Space Administration (NASA; Johnson Space Center). This study was conducted in accordance with the Declaration of Helsinki.

Exercise Countermeasure

The pre- and in-flight exercise programs of each crew member have been published elsewhere (26), and, due to their importance to this work, are summarized in Table 1. The exercise profiles were determined from crew member logbooks and from downloaded analog data from the treadmill and cycle ergometer. The exercise countermeasures, including the resistance exercise, were classified as moderate intensity aerobic activities. In our previous publication (11), we found that the degree of treadmill running influenced the extent of fiber atrophy. Less atrophy occurred as total treadmill running in minutes per week increased (r = 0.68). Thus we subdivided the crew into those running 200 min/wk or more (high treadmill, HT) from those running <100 min/wk (low treadmill, LT). The weekly treadmill running average for each crew member has been published (26).

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Table 1.

Summary of aerobic and resistance exercise performed while on the ISS

Muscle Biopsies

A muscle biopsy of ∼80 mg was obtained from the mid-belly of the lateral head of the gastrocnemius and soleus muscles of each crew member prior to launch (L-55 ± 2) and on landing day (R + 0) as described previously (11, 26). The biopsy sample was divided longitudinally into three portions for structural, biochemical, and functional studies, with the latter reported on previously (11). Two of the portions were longitudinally aligned on small index cards and frozen in liquid nitrogen. These samples were shipped in a liquid nitrogen dry shipper to Marquette University. One portion was used for histochemical and immunostaining studies of enzymes and substrates, and the other was freeze-dried under vacuum at −35°C and stored under vacuum at −80°C for single fiber biochemical analyses of selected metabolic enzymes (9).

Histological Assays

Tissue sectioning and staining.

Serial cross-sections (8–10 μm) of muscle were cut with a cryostat microtome and picked up on glass slides. Histochemical fiber typing was performed by reacting for actomyosin ATPase activity (acid and alkaline preincubation) and immunostaining for myosin heavy chain specific antibodies for type I, IIa, and IIx myosin (12). Immunospecific protein staining was also performed for citrate synthase, carnitine palmitoyltransferase, phosphofructokinase, glucose transporter type 4, and uncoupling protein 3. No marked differences in these proteins were observed subjectively for preflight and postflight muscles, so antibody staining quantitation was not conducted. Mitochondrial oxidative activity was demonstrated by reacting for cytochrome oxidase activity (30). Oil Red O staining was used to assess intracellular lipid content (17). Glycogen content was visualized by periodic acid Schiff (PAS) staining (22). Sections of the preflight and postflight muscles were collected on the same slide so that the conditions of staining were identical.

Fiber type percentage, size determination, and assessment of staining.

In acid-preincubated ATPase sections, the percentages of darkly stained slow, lightly stained fast, and intermediately reactive hybrid fibers were counted, and fiber type sizes were measured using computerized morphometry (Metamorph 4.6). Cytochrome oxidase reaction product content was measured per muscle fiber using the thresholding detection algorithm in Metamorph and normalized to fiber cross-section area as area percent staining. Lipid content per fiber was quantified by thresholding detection of Oil Red O stained droplets and normalizing to fiber area. The PAS reaction for glycogen produced a pink coloration of the fiber. The glycogen content was defined as the optical density of the staining product within single muscle fibers using Metamorph morphometry and densitometry algorithms.

Single Fiber Biochemical Analysis of Aerobic and Anaerobic Enzymes

Fiber preparation.

The freeze-dried samples were warmed to room temperature while under vacuum, and then fibers (∼2 mm long) were dissected free in a temperature- and humidity-controlled room (22°C, < 30% humidity). The isolated fibers were restored individually under vacuum at −80°C. At the time of assay, the fibers were returned to room ambient conditions and divided into three equal segments. Two segments were used for the enzyme assays described later, and the third was run on SDS-PAGE for fiber-type determination by myosin heavy chain content as described previously (11, 29). Each segment was weighed on a quartz fiber balance (19).The assays were conducted as described by Lowry and Passonneau (19), using methods based on pyridine nucleotide enzyme reactions. In all cases, the sample mass was large enough to allow direct measurement of the formed pyridine nucleotide. The enzyme activities were expressed as moles per kilogram dry weight per hour. The assays described below were carried out in 1.5-ml centrifuge tubes.

Citrate synthase.

Individual freeze-dried fiber segments (300–400 ng) from the soleus and gastrocnemius were dissolved (10 ng/μl) in a solution containing 20 mM phosphate buffer (pH 7.4), 0.6 M KCl, 0.05% BSA, 0.5 mM EDTA, 5 mM β-mercaptoethanol, and 0.5% Triton X-100 and preincubated at 25°C for 2 h. After preincubation, 2 μl were transferred to 25 μl of specific reagent and incubated for 60 min at 25°C. The specific reagent contained 50 mM Tris-HCl buffer (pH 8.1), 0.25% BSA, 0.5 mM oxaloacetate, and 0.5 mM acetyl coenzyme A. The reaction was stopped by adding 10 μl of 0.2 M NaOH and heating at 95°C for 5 min. After cooling to 25°C, 250 μl of the citrate reagent were added, and the mixture was incubated in the dark at 25°C. The citrate reagent contained 50 mM Tris-HCl (10:40), 0.1 mM ZnCl2, 0.02% BSA, 0.1 mM NADH, 2.5 U/ml malic dehydrogenase, and 0.024 U/ml citrate lyase. After 25 min, the excess NADH was destroyed by adding 30 μl of 1 M HCl and incubating for 20 min at 25°C. A 100-μl aliquot of the reaction assay was added to 1 ml of 6 N NaOH plus 10 mM imidazole and heated in the dark for 20 min at 60°C to destroy the NAD and to produce a fluorescent product. The reaction was cooled to 25°C, and the fluorescence intensity was measured.

β-Hydroxyacyl-CoA dehydrogenase.

After preincubation (as described for citrate synthase earlier), 2 μl were transferred to 100 μl of specific reagent and incubated in the dark for 60 min at 25°C. The specific reagent contained 150 mM imidazole buffer (pH 7.0), 0.6 M HCl, 1 mM EDTA, 0.05% BSA, 125 μM NADH, and 1 mM acetoacetyl CoA. The reaction was stopped by adding 10 μl of 0.5 N HCl and holding at 25°C for 10 min. The NAD produced was converted to a fluorescent product as described for the citrate synthase assay, and the fluorescence was measured.

Lactic dehydrogenase.

Fiber preincubation, reaction sequence (including aliquot volumes and incubation times), and production and measurement of the fluorescent product were the same as described for the β-hydroxyacyl-CoA dehydrogenase assay, except the reaction was stopped with 10 μl of 1 N HCl. The specific reagent contained 100 mM imidazole buffer (pH 7.0), 40 mM sodium pyruvate, 0.05% BSA, and 125 μM NADH.

Phosphofructokinase.

The fiber segment was added directly to 100 μl of specific reagent and 10 μl of H2O, and the assay was conducted as described above for lactic dehydrogenase. The specific reagent was comprised of 50 mM Tris-HCl (pH 8.1), 10 mM K2HPO4, 1 mM 5′AMP, 1 mM ATP, 1.5 mM fructose-6-phosphate, 0.05% BSA, 1 mM MgCl2, 2 mM NADH, 0.2 U/ml aldolase, 20 U/ml triose phosphate isomerase, and 3 U/ml α-glycerolphosphate dehydrogenase.

Statistical Analyses

For histochemical analyses and biochemical determinations of aerobic and anaerobic enzyme activities in single slow type I and fast type II fibers, statistical differences between the pre- and postflight values for each crew member and for the group means (LT vs. HT groups) were assessed using Student's unpaired two-tailed t-test with a 0.05 level of confidence.

RESULTS

The effect of prolonged microgravity on slow type I and fast type II fiber size was reported in detail previously (11). Here our purpose was to relate the atrophy of a given fiber type to specific enzyme and substrate changes. As reported previously (11), the degree of fiber atrophy varied greatly between crew members, and considerable variability was also found in muscle enzymes. The LT crew members, running <100 min/wk, showed more fiber atrophy than the HT group, running 200 min/wk or more (11). For the soleus muscle, the average decline in diameter for the type I and II fibers was 20 and 16%, respectively, but the differences between the LT and HT groups was substantial for the soleus type I (29% LT vs. 8% HT), and type II (27% LT vs. 3% HT) fibers (11). Subjects were identified with the letters A to I using the same letter scheme as in our previous publications (11, 26). Subjects B, E, G, and H are in the HT group, and Subjects A, C, D, F, and I are LT members.

Soleus Muscle Histochemistry: Enzyme Activity and Substrate Content

Figures 1 and 2 show cross-sections of pre- and postflight soleus muscle fibers reacted for actomyosin ATPase activity to illustrate fiber types in LT Subject C (Fig. 1, A and B) and HT Subject E (Fig. 2, A and B). In the acid-preincubated ATPase sections, type I slow-twitch oxidative fibers are darkly stained. Type IIa fast-twitch oxidative glycolytic fibers are lightly reactive. Based on immunostaining for fast and slow myosins (data not shown), the moderately reactive fibers are hybrid fibers containing both fast and slow myosin (Fig. 2A). Histologically, there were insufficient numbers of IIx fibers in the soleus and gastrocnemius muscle sections to quantify cytochrome oxidase (CO), lipid, or glycogen content in this fiber type, so the measurements in Tables 2, 3, and 4 were restricted to type I and IIa fibers. Postflight atrophy is evident in the fibers of LT Subject C (Fig. 1B), whereas atrophy did not occur in the HT Subject E (Fig. 2B).

Fig. 1.

Soleus. Low treadmill (LT) Subject C preflight (A, C, E) and postflight (B, D, F) muscle serial sections are stained for acid-preincubation actomyosin ATPase activity (A, B), cytochrome oxidase activity (C, D), and Oil Red O for intramyofiber lipids (E, F). Type I fibers are dark and type IIa fibers are light in the ATPase sections. Postflight fibers exhibit atrophy. Cytochrome oxidase activity and lipid content are decreased postflight. This subject's changes are representative of the LT group. Bar = 90 μm.

Fig. 2.

Soleus. High treadmill (HT) Subject E preflight (A, C, E) and postflight (B, D, F) muscle serial sections are stained for acid-preincubation actomyosin ATPase activity (A, B), cytochrome oxidase activity (C, D), and Oil Red O for intramyofiber lipids (E, F). Type I fibers are dark, hybrid fibers are moderate, and type IIa fibers are light in the ATPase sections. Postflight fibers are not atrophied. Cytochrome oxidase activity (D) and lipid content (F) in postflight fibers are at or above preflight levels. Bar = 90 μm.

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Table 2.

Cytochrome oxidase

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Table 3.

Histological determination of lipid and glycogen content in slow type I and fast type IIa soleus fibers pre- and postflight

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Table 4.

Histological determination of lipid and glycogen content in gastrocnemius type I and fast type IIa fibers pre- and postflight

In the preflight samples of all subjects, the mean CO activity for soleus type IIa fibers was significantly less than for the slow type I fibers. Postflight, the all crew member mean for CO activity for the fast type IIa fiber was unchanged, while the activity of this enzyme was significantly depressed in the slow type I fiber (Table 2). Despite no change in the composite mean (HT and LT groups combined), soleus type IIa fibers of the HT group had postflight values significantly higher than preflight, while the LT group mean was unaltered (Table 2). The CO activities for Subjects C and E are shown in serial section (Fig. 1, C and D, and Fig. 2, C and D, respectively). The CO reaction product indicates an abundance of mitochondria in subsarcolemmal clusters and within myofibrils. The area percent of CO reaction product per muscle fiber area was reduced by 64% in the postflight soleus of Subject C (Fig. 1, C and D; Table 2). The average reduction was 59% for the four LT subjects (Table 2). For Subject E, the CO activity was not significantly altered postflight (Fig. 2, C and D; Table 2). The three HT subjects on average showed no significant change in CO activity, but large individual variations were present in soleus type I fiber, with Subject B highly increased, Subject E unchanged, and Subject H significantly down (Table 2).

Soleus fiber lipid content was quantified in Oil Red O stained sections (Fig. 1, E and F, and Fig. 2, E and F). The lipid-stained fibers are serial to the acid ATPase sections. The lipid content preflight was higher in type I fibers than IIa fibers (Table 3). Postflight in the LT group, there was loss of lipid in type I fibers, while the lipid content was preserved in the HT group (Table 3). Pooling All crew members, the soleus type I fiber lipid was unchanged postflight (Table 3). Representative of the LT group, Subject C's soleus type I fibers exhibited decreased lipid postflight (Fig. 1, E and F). In contrast, HT Subject E had higher type I fiber lipid preflight than Subject C, and the lipid content was maintained postflight (Fig. 2, E and F; Table 3).

Gastrocnemius muscle histology.

Atrophy was pronounced in the gastrocnemius type I and II fibers in the LT group. Figure 3 illustrates fiber atrophy in alkaline-preincubation actomyosin reacted sections from Subject D (Fig. 3, A and B). This individual exhibits a shift from mostly type I fibers (lightly stained) to expression of darkly stained type II fibers postflight (Fig. 3B). Immunostaining with myosin isoform specific antibodies for type IIa and IIx revealed that a small proportion of type II fibers contained IIx myosin (data not shown). Serial sections revealed that CO decreased postflight in large part due to loss of peripheral subsarcolemmal staining (Fig. 3, C and D). CO levels were significantly lower in gastrocnemius type I fibers of the LT group postflight (Table 2). For the LT and HT groups, lipid concentration in gastrocnemius type I and IIa fibers tended higher postflight but not significantly (Table 4). However, a significant increase in lipid content was observed postflight for Subject D and Subject E for the type I fiber, and Subject H for the type IIa fiber (Fig. 3, E and F, Table 4).

Fig. 3.

Gastrocnemius. LT Subject D preflight (A, C, E) and postflight (B, D, F) serial sections stained for alkaline-preincubation actomyosin ATPase activity (A, B), cytochrome oxidase activity (C, D), and Oil Red O for intramyofiber lipids (E, F). Type I fibers are light and type IIa fibers are dark in the ATPase sections. Postflight atrophy of type I and type IIa fibers is present. Cytochrome oxidase activity is decreased postflight, especially in the peripheral subsarcolemmal regions. Lipid staining is increased postflight for this subject, but lipid content was generally not different from preflight for most subjects. Bar = 75 μm.

Fiber glycogen.

Fiber glycogen was assessed histologically with PAS staining. Postflight soleus glycogen was significantly elevated for slow type I and fast type IIa fibers when all crew were compared quantitatively for PAS staining by optical density (Table 3). Representative micrographs illustrate LT Subject C soleus in which glycogen significantly increased postflight in both type I and IIa fibers (Fig. 4, A and B). For the gastrocnemius muscle, the preflight levels of glycogen in type I and IIa fibers are higher than that in soleus (Tables 3 and 4), and all crew quantitation reveals maintenance of PAS staining in the postflight fibers (Table 4). When the individual changes are examined, there are significant increases as shown in Fig. 4, C and D, for Subject A, and decreases in gastrocnemius glycogen staining that are not explained by HT or LT exercise (Table 4).

Fig. 4.

Soleus (A, B) and gastrocnemius (C, D) fibers stained with periodic acid Schiff reagent for glycogen. In the LT Subject C preflight soleus section (A), type I fibers are light to moderate and type IIa fibers are dark. Postflight (B) staining is increased. Elevated glycogen is typical for soleus fibers in the LT and HT subjects. The gastrocnemius fibers of LT Subject A show increased levels of glycogen postflight (D). For gastrocnemius, Subjects A, E, and I had elevated glycogen, whereas glycogen was decreased in Subjects C, D, and H. Bar = 60 μm.

Biochemical Analysis of Enzyme Capacity

Soleus muscle.

The effect of prolonged spaceflight on the glycolytic enzymes LDH and phosphofructokinase, and the mitochondrial enzymes β-hydroxyacyl-CoA dehydrogenase (βOAC) and citrate synthase (CS), in soleus type I and type II fibers is shown in Tables 5 and 6, respectively. When all crew members were grouped together, there was no change in the soleus type I fiber glycolytic or mitochondrial enzyme activity (Table 5). However, two of the three HT (Subjects E and H) showed significant increases in both βOAC and CS in the soleus type I fibers (Table 5). One LT, Subject I, also showed an increased mitochondrial enzyme capacity, but the magnitude of the increase (11% and 25%) was considerably less than that for the HT subjects where the increase ranged from 40% to 87%. Regarding the soleus type I fiber, Subject E (HT group) exhibited the largest increase in oxidative enzymes and was the only subject to show a significant decrease in glycolytic enzyme activity. In contrast, Subject C (LT group) showed a large decline in cytochrome oxidase and a significant increase in the glycolytic enzyme LDH (Table 5). Figure 5 shows a plot of CS vs. LDH for Subjects C and E for those fibers in which the activity of these two enzymes were determined. The plot demonstrates the rather wide range of enzyme activities that exist within a given fiber type, the postflight shift to higher aerobic (CS) and lower glycolytic (LDH) enzyme activities in HT Subject E, and the elevated LDH in LT Subject C (Fig. 5).

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Table 5.

Soleus type I fiber glycolytic and oxidative enzyme activity pre- and postflight

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Table 6.

Soleus type II fiber glycolytic and oxidative enzyme activity pre- and postflight

Fig. 5.

Comparison of citrate synthase (CS) and lactate dehydrogenase (LDH) activities in single slow type I fibers isolated from the soleus of Crew Member C (LT group) and Crew Member E (HT group). Each symbol represents a single fiber with filled symbols preflight and open symbols postflight. The fibers from a given muscle sample are surrounded by a solid (preflight) or a dotted (postflight) line. Postflight Subject E's fibers showed greater oxidative enzyme activity, while Subject C's fibers showed greater glycolytic enzyme activity compared with the preflight condition.

Glycolytic and mitochondrial enzyme activities of fast type II fibers in the soleus were, with few exceptions, unaltered by the 6 mo of microgravity. The exception was LDH, where Subjects C and H showed a significant decrease and Subject D a significant increase postflight (Table 6).

Gastrocnemius muscle.

The glycolytic and mitochondrial enzyme activities of the gastrocnemius slow type I and fast type II fibers, and their changes with prolonged space flight, are shown in Tables 7 and 8. From a qualitative perspective, the microgravity effects in the slow type I fiber were similar to those observed for this fiber type in the soleus, in that no changes were observed in group means for any enzyme. Examination of the individuals revealed that subjects who showed a significant increase in CS activity (Subjects A, E, and G) showed a significant decrease in LDH activity, and Subject I with a postflight decline in CS showed increased LDH (Table 7). Figure 6 illustrates this point by showing a plot for βOAC vs. LDH for gastrocnemius type I fibers for Subjects D and G pre- and postflight. Similar to the soleus, within a fiber type there is a wide range of activities for a given enzyme, and Subject G with a significant postflight increase in βOAC showed a significant decline in LDH activity. In comparison, Subject D showed a significant increase in LDH, which was accompanied by a small but nonsignificant decline in βOAC.

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Table 7.

Gastrocnemius type I fiber glycolytic and oxidative enzyme activity pre- and postflight

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Table 8.

Gastrocnemius type II fiber glycolytic and oxidative enzyme activity pre- and postflight

Fig. 6.

Comparison of β-hydroxyacyl-CoA dehydrogenase (βOAC) and LDH activities in single slow type I fibers isolated from the gastrocnemius of Crew Member D (LT group) and Crew Member G (HT group). Each symbol represents a single fiber, with filled symbols preflight and open symbols postflight. The fibers from a given muscle sample are surrounded by a solid (preflight) or a dotted (postflight) line. Postflight Subject G's fibers showed greater oxidative enzyme activity and reduced glycolytic capacity, while Subject D's fibers showed greater glycolytic enzyme activity compared with the preflight condition.

The gastrocnemius type II fiber showed no group mean changes with microgravity for any of the enzymes studied (Table 8). However, similar to the type I fiber, when a significant increase in oxidative enzyme was observed, as for Subject H, a significant decline was observed for LDH (Table 8). Comparing the LT and HT group means, one sees that there were no significant differences pre- vs. postflight, but βOAC tended up and LDH tended down in the HT group, with the opposite observed for the LT group (Table 8). The reciprocal change for the HT group, with the glycolytic enzyme decreasing and the oxidative enzymes increasing postflight, is shown in Fig. 7 for Subject H. Figure 7 also shows the postflight increase in LDH for LT Subject A.

Fig. 7.

Comparison of βOAC and LDH activities in single fast type II fibers isolated from the gastrocnemius of Crew Member A (LT group) and Crew Member H (HT group). Each symbol represents a single fiber, with filled symbols preflight and open symbols postflight. The fibers from a given muscle sample are surrounded by a solid (preflight) or a dotted (postflight) line. Postflight Subject H's fibers showed greater oxidative enzyme activity and reduced glycolytic capacity, while Subject A's fibers showed greater glycolytic enzyme activity compared with the preflight condition.

DISCUSSION

The results presented in this study are the first to evaluate the effects of prolonged space travel (weightlessness plus exercise countermeasures) on the activity of metabolic enzymes and muscle substrates. We have shown previously that, despite in-flight aerobic and strength exercise-training, prolonged weightlessness caused considerable declines in fiber mass, force, and power, with the greatest effects observed in slow type I fibers of the soleus (11, 26). We observed that these changes were partially attenuated by treadmill running; as a result, here and in our previous publications (11, 26), the crew members were subdivided into those running 200 min/wk or more (HT) and those running <100 min/wk (LT).

A consistent observation is that crew members during weightlessness experience increased fatigue, and this is particularly true during extravehicular activities (5, 18). This is in part explained by declines in peak aerobic capacity and muscle atrophy, so that loads represent a higher percentage of the crew member's peak aerobic capacity (11, 26). The extent to which the latter could be attributed to declines in stroke volume and thus cardiac output vs. a reduced tissue oxidative capacity is unknown. With short-duration space flight, we observed that V̇o2 elicited at a HR equal to 85% of the crew members' preflight maximum HR showed a rapid and continued decline between day 2 (−6.2%) and day 13 (−11.3%), and recovered rapidly postflight (27). Others have shown that short-duration space flight reduces mitochondrial protein, but the decline is less than fiber atrophy, so the oxidative enzyme activity remained unaltered or slightly elevated when activity was expressed per gram of dry weight (8, 10, 20).

Muscle Lipids and Glycogen

We previously published that LT crew members showed at the electron microscopic level a high degree of slow fiber atrophy in soleus muscle and fewer intracellular lipids (11). Our current results confirmed this observation, because, based on Oil Red O staining, soleus type I fiber lipid was significantly reduced in the LT but not the HT crew members. This observation is opposite to that seen with short-duration space flight where fiber lipid was increased in humans and rats (24, 28). It is not clear why intracellular lipid depletion is associated with fiber atrophy; however, one possibility includes an increased reliance on the oxidation of intramuscular lipids as an energy source. Alternatively, intracellular fat depletion in the LT group could have been mediated by a reduced free fatty acid (FFA) mobilization and delivery to the muscle. In support of this hypothesis, we previously observed that 15 days of rat hindlimb suspension (a model of weightlessness) attenuates the redistribution of visceral blood flow normally observed during exercise (21). Although blood flow distribution during exercise in space has not been studied, the chronic stress and elevated blood catecholamine levels associated with prolonged space flight might decrease the cellular response to sympathetic drive (21, 31). This in turn could inhibit the mobilization of FFAs and reduce the redistribution of blood flow from the viscera to the working skeletal muscles, thus decreasing FFA delivery, uptake, and use by the muscle. In this study, we found no depression of βOAC, a marker enzyme of the β-oxidation pathway. In fact, this enzyme showed a postflight increase in the soleus type I fibers in three of the crew members. Following a 9-day space flight, Baldwin et. al. (1) made a similar observation that enzymes of the β-oxidative pathway were unaltered in rat hindlimb muscle. However, they observed a significantly reduced ability of the rat vastus muscle to oxidize long-chain fatty acids, and they hypothesized that the inhibition of fat oxidation may have resulted from a reduced ability to activate or translocate fats into the mitochondria. The extent to which fat oxidation may be limited by FFA delivery and/or translocation of fats into the mitochondria in humans awaits in-flight exercise and muscle biopsy studies.

As expected, preflight glycogen content was higher in the fast type IIa than the slow type I fibers, and this was true in the soleus and gastrocnemius. The highest content was observed in the fast fibers of the gastrocnemius. Postflight, the glycogen content was significantly increased in the fast and slow fibers of the soleus, but no change was observed in the gastrocnemius. It is not possible to determine whether the increase was caused by weightlessness or the fact that the crew consumed carbohydrate-rich foods 12 h before return to earth. The observation that the glycogen increase was restricted to the soleus could be explained by a supercompensation mechanism (13). If the glycogen content of the soleus was chronically low during weightlessness, the carbohydrate loading before return to earth may have stimulated glycogen synthase, causing glycogen supercompensation (2, 14).

Muscle Enzymes

Consistent with previous observations for humans, the subjects in this study showed a rather wide range of activities for a given enzyme within a particular fiber type and muscle (4, 10, 15). For example, the soleus type I fiber CS activity for Subject C preflight averaged 6.31 ± 0.36 (Table 5), but the range of activity was 3.03 to 8.75 mol·kg−1·h−1 (Fig. 5). A second observation was that the difference in oxidative potential was only slightly higher in type I compared with type II fibers. This was true for both the Krebs cycle enzyme CS and the β-oxidation pathway enzyme βOAC. In part, this resulted from the fact that most of the fast fibers in both muscles assayed were type IIa. Preflight, none of the crew members contained any fast type IIx fibers in their soleus muscles, and postflight this population averaged only 1% (26). In the gastrocnemius, the pure type IIx population averaged 3% in both pre- and postflight samples (26). In contrast to aerobic enzymes, reasonably large differences were observed in the glycolytic marker enzymes between fast and slow fibers, with the fast fibers showing considerably higher activity (Tables 58).

Based on the larger fiber size and higher aerobic enzyme activities preflight, the LT group entered the flight in better condition, but their in-flight training program was less effective compared with the HT group. Consequently, postflight the soleus type I fiber aerobic enzyme activities (both CS and βOAC) were higher in the HT group compared with the LT group. As reported previously, the high-volume, moderate-intensity resistance exercise program was insufficient to prevent fiber atrophy (11). However, treadmill running did appear to partially protect against fiber atrophy and, in subjects who ran 200 min/wk or more (HT group), helped to increase muscle aerobic enzyme activity. This observation is important, because it demonstrates that in-flight programs of exercise training of the appropriate nature (i.e., mode, duration, intensity) cannot only prevent loss, but can stimulate significant increases in muscle aerobic enzyme activity. It does not appear that the increased fatigue during and following weightlessness experienced by astronauts and cosmonauts can be directly attributed to a loss of muscle aerobic enzyme capacity. In this study, even the LT group maintained unaltered or slightly elevated aerobic muscle enzyme activity. However, because muscle mass was significantly reduced (∼15% for the soleus) following the 6 mo of weightlessness, there would be less enzyme (i.e., mitochondria) and contractile protein in absolute terms. Thus, when extrapolating to whole body activities such as extravehicular activities or exercise, there would be a greater relative demand for the same workload due to less muscle mass. This would be both metabolic and fiber performance (i.e., power) issue which, combined, emphasizes the importance of maintaining the metabolic health of the crew by preventing the loss of muscle mass during prolonged periods in microgravity.

In conclusion, the results of this study demonstrate that, despite prolonged weightlessness, crew members are able to maintain their muscle aerobic and glycolytic enzyme capacity and that, with adequate amounts of treadmill running, even are able to improve muscle oxidative capacity. The decline in type I fiber intracellular lipid in the antigravity soleus muscle was correlated with fiber atrophy, and was prevented by treadmill running that exceeded 200 min/wk. The increased fatigability reported to occur with weightlessness cannot be directly attributed to a loss of muscle substrate or enzyme capacity, but likely is in part due to fiber atrophy, such that extravehicular activities require greater fiber recruitment, both an increased number of fibers and activation frequency, both of which lead to an earlier onset of fatigue.

GRANTS

This research was supported by NASA Grant NCC9-116 to R. H. Fitts.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: R.H.F., S.W.T., D.L.C., and D.A.R. conception and design of research; R.H.F., P.A.C., J.L.B., and D.A.R. performed experiments; R.H.F., J.L.B., and D.A.R. analyzed data; R.H.F., S.W.T., D.L.C., J.L.B., and D.A.R. interpreted results of experiments; R.H.F., P.A.C., J.L.B., and D.A.R. prepared figures; R.H.F. and D.A.R. drafted manuscript; R.H.F., S.W.T., D.L.C., J.L.B., and D.A.R. edited and revised manuscript; R.H.F., S.W.T., D.L.C., and D.A.R. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank Jim Peters, who worked closely with the crew, and the astronauts and cosmonauts who graciously gave their time and energy to this research. We thank Simone Thomas, Elkin Romero, Alicia Forrester, the flight surgeons, and the numerous unnamed people who assisted our research team at NASA and the Russian Space Agency.

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