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J Appl Physiol 92: 817-825, 2002; doi:10.1152/japplphysiol.00717.2001
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Vol. 92, Issue 2, 817-825, February 2002

Thin filament diversity and physiological properties of fast and slow fiber types in astronaut leg muscles

Danny A. Riley1, James L. W. Bain1, Joyce L. Thompson1, Robert H. Fitts2, Jeffrey J. Widrick2, Scott W. Trappe3, Todd A. Trappe3, and David L. Costill3

1 Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee 53226; 2 Department of Biology, Marquette University, Milwaukee, Wisconsin 53201; and 3 Human Performance Laboratory, Ball State University, Muncie, Indiana 47306


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Slow type I fibers in soleus and fast white (IIa/IIx, IIx), fast red (IIa), and slow red (I) fibers in gastrocnemius were examined electron microscopically and physiologically from pre- and postflight biopsies of four astronauts from the 17-day, Life and Microgravity Sciences Spacelab Shuttle Transport System-78 mission. At 2.5-µm sarcomere length, thick filament density is ~1,012 filaments/µm2 in all fiber types and unchanged by spaceflight. In preflight aldehyde-fixed biopsies, gastrocnemius fibers possess higher percentages (~23%) of short thin filaments than soleus (9%). In type I fibers, spaceflight increases short, thin filament content from 9 to 24% in soleus and from 26 to 31% in gastrocnemius. Thick and thin filament spacing is wider at short sarcomere lengths. The Z-band lattice is also expanded, except for soleus type I fibers with presumably stiffer Z bands. Thin filament packing density correlates directly with specific tension for gastrocnemius fibers but not soleus. Thin filament density is inversely related to shortening velocity in all fibers. Thin filament structural variation contributes to the functional diversity of normal and spaceflight-unloaded muscles.

human; skeletal muscle; electron microscopy; myofilaments; actin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DURING SPACEFLIGHT, HUMANS can undergo detrimental biochemical, physiological, and structural changes in fast and slow skeletal muscle fibers (6, 7, 23, 29, 33-35). Skeletal muscle weakness, fatigue, incoordination, and delayed-onset muscle soreness are evident on return to gravity loading (6, 7). The muscle soreness, which occurs without strenuous exercise, indicates increased susceptibility of atrophic muscle fibers to weight-bearing-induced damage (7, 14, 36). The changes induced by spaceflight and a variety of ground-based unloading models can involve skeletal muscles in general, appear specific for fast or slow muscle, or selectively target fast or slow muscle fiber types (1, 5-7). Determining whether particular structural and functional properties are regulated at a general, muscle type-, or muscle fiber-type level improves the understanding of the mechanistic basis for the adaptive or degenerative changes and offers insights into possible countermeasures.

Skeletal muscle mass decreases during extended unloading. In rats, slow muscles are more affected than fast muscles and slow fibers more than fast fibers, but this hierarchy does not always apply in human muscles (7). Muscle mass primarily reflects the amount of contractile proteins, which are manifested ultrastructurally as thick (primarily myosin) and thin (primarily actin) filaments. Our laboratory has shown that, when myofilaments are lost in soleus type I fibers during bed rest and spaceflight, the ratio of actin to myosin falls, and thin filament packing density is disproportionately decreased relative to thick filament density. The resulting reduced packing density appears to increase the velocity of shortening in the absence of fast myosin expression (22, 23, 33-35). The accelerated loss of thin filaments, i.e., decreased thin filament density within the overlap region of the A band, results from an increase in both short filaments and missing filaments (22, 23). After spaceflight and bed rest, specific tension only decreased 10% or less, which meant greater tension loading per remaining thin filament and possibly increased susceptibility to sarcomere structural failure during reloading (33-35). These studies emphasize the importance of understanding the regulation of thin filament structure in muscle fiber types because of the profound effects of thin filament properties on muscle function.

The present study examined gastrocnemius and soleus muscle fibers obtained from astronauts participating in the 17-day, Life and Microgravity Sciences Spacelab (LMS) Space Shuttle Transport (STS-78) mission. Myofilament structural properties were correlated with biochemical and contractile physiological properties of gastrocnemius type I, IIa, and IIa/IIx fibers and soleus type I fibers obtained before (normal) and after (unloaded) LMS spaceflight. The findings demonstrate that there is remarkable diversity of thin filament architecture across muscles and fiber types both before and after spaceflight, which influences physiological properties such as velocity of shortening and specific tension.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Test subjects. Gastrocnemius and soleus muscles of four male astronauts, averaging 43 ± 4 yr old, 183 ± 8 cm tall, and 86 ± 6 kg body wt before flight, were biopsied before spaceflight and after landing following the 17-day National Aeronautics and Space Administration LMS STS-78 mission (June 1996). The soleus muscle fibers, described in a previous report (23), were analyzed in additional ways for this comparative study. Subjects were designated astronauts A-D. An open-incision, needle biopsy was obtained 45 days before launch from the left muscles of each individual as a preflight control. Within 3 h after landing, a postflight biopsy was removed from the right muscles. Physical activity was restricted postflight by seating the subjects in wheelchairs until biopsy because return to weight bearing can induce secondary degenerative changes in atrophic muscles (14, 36). Before (90, 60, 30, and 15 days before launch), during (day 2 or 3, day 8 or 9, and day 12 or 13), and after flight (2 and 8 days after landing), subjects underwent physiological testing using an isokinetic dynamometer and cycle ergometry (30). Each testing session consisted of determination of isometric and isokinetic torque of the right ankle extensors and incremental supine cycle ergometry performed at work rates up to 85% of preflight maximal oxygen uptake (30, 34, 35). Individuals performed ad libitum aerobic exercise inflight, which was not possible to document. The human use protocol was approved by the Institutional Review Boards of the participating institutions, and informed written consent was obtained before participation.

Tissue processing. Each biopsy was subdivided into bundles for single-fiber contractile physiology and biochemistry, histochemistry, and electron microscopy. The portion for physiology was placed in cold (4°C) skinning solution and shipped to Marquette University for measurement of shortening velocity and specific tension and for gel electrophoresis for resolution of myosin composition as performed previously; details are described in the companion papers (33-35). Another portion was pinned out straight to a flat plastic stick and fixed in 20 ml of 4% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.2) with 5 mM CaCl2 for 2 h at room temperature followed by 24-48 h at 4°C. The fixed tissues were shipped overnight at 4°C to the Medical College of Wisconsin for completion of fixation in 1.3% OsO4. Longitudinal and cross thin sections (70 nm) were cut and poststained with uranyl acetate and lead citrate before examination and photographing in a JEOL 100 CXII electron microscope. Morphological changes induced by spaceflight were identified by comparing the ultrastructural features of the postflight test samples with those of the preflight control tissue from the same individual. The means of the pre- and postflight measures were analyzed by a paired-sample t-test analysis with each subject serving as his own control. Bed rest and spaceflight data supported an a priori prediction of decreased thin filament densities in the postflight condition and permitted a one-tailed t-test.

Quantitation of thick and thin filament density. Gastrocnemius muscle fibers were typed in longitudinal electron microscopic sections as slow red, fast red, and fast white fibers on the basis of published morphological criteria for human muscles (Fig. 1, A and B) (2, 26). The aldehyde-fixed fibers could not be immunostained for myosin isoform typing. However, the research literature on fiber-type ultrastructure, histochemical myosin ATPase activity, isomyosin immunostaining, and single-fiber electrophorectic resolution of myosin types permitted matching of slow red to type I, fast red to type IIa, and fast white to types IIa/IIx and IIx fibers (1, 25, 26). The myofilament density measurements were related to sarcomere length of the same fibers and to the physiological and biochemical properties of type I, IIa, and IIa/IIx fibers from the same biopsies. Previous fiber typing of these biopsies by myosin heavy chain gel patterns revealed that 93% of the fast white fibers were IIa/IIx and that 7% were IIx (35). It was not possible in the present study to ascertain electron microscopically the proportions of IIa and IIx myosins in the 40 fast white fibers examined, but the 7% occurrence of pure IIx fibers indicates that 3 of 40 sampled may be type IIx. For simplicity, in this paper the type IIa/IIx classification includes type IIa/IIx and type IIx fibers.


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  		Fig. 1.   Type IIa/IIx fast white, type IIa fast red, and type I slow red fiber types are distinguishable in longitudinal electron micrographic sections of preflight control gastrocnemius muscles. A: type IIa/IIx fibers possess narrow (52 ± 2 nm) Z bands and a paucity of mitochondria. B: type I fibers have very wide (101 ± 3 nm) Z bands, abundant large mitochondria (M), and numerous lipid droplets (L). Type IIa fibers exhibit wide (73 ± 7 nm) Z bands and abundant mitochondria. Z-band widths were measured for 25 fibers of each type. The distinguishing features of fiber types persisted after spaceflight. Bar = 1.5 µm.

Sarcomere lengths for individual fibers were measured in longitudinal sections, and the typed fibers were reoriented and cross-sectioned for filament density measurements as performed previously (22, 23). For the 160 aldehyde-fixed fibers studied, sarcomere lengths ranged from 1.83 to 3.37 µm. As described previously, myofilament density is directly proportional to sarcomere length (22). To compare filament density in the A band across fibers, the packing densities (filament number per unit area) of thick and thin filaments were normalized to 2.5 µm by the following calculation: density × (actual sarcomere length divide  2.5). For each of the four astronauts, five fibers of each of three fiber types were assayed morphologically for each of two conditions (pre- and postflight). A total of 120 fibers was analyzed from the gastrocnemius muscles. For soleus, a total of 40 type I fibers were examined. Data are reported as means ± SE.

Thick and thin filament concentrations were assessed in the overlap A-band region, i.e., the region in which thin filaments overlap with thick filaments near the A band- I band boundary (Fig. 2A). Micrographs were overlaid with a transparency of grid squares of 0.0056 µm2 at ×201,000. The number of filaments counted per 0.0056 µm2 was multiplied by 178.57 to compute filament number per square micrometer. These density values of thick filaments were used to derive average spacing in nanometers by multiplying the inverse square root of the density by 1,000. This conversion assumes a uniform spacing of thick filaments in the A bands, which was consistent with their appearance in the electron micrographs. For nonbiased sampling, the grid squares were positioned at random in the sampling regions of centrally located myofibrils. The peripheralmost myofibrils were avoided because sarcomere structure was normally variable and sometimes incomplete, especially near myonuclei. To avoid biasing filament counts downward, grid placement was shifted when either the myofilaments were not oriented in cross section, appearing as lines instead of dots, or the predominant feature was a cluster of glycogen particles (Fig. 2B).


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  		Fig. 2.   Different levels of sectioning of myofibrils are shown for postflight soleus type I fibers. A: thick and thin filament densities were measured in the overlap A band (OA) region. The M line (M) is devoid of thin filaments. B: thin filament densities were also measured within the I band (I) ~150 nm from the Z band (Z) where thin filaments retain a square-array distribution. Z-band lattice spacing was determined by measuring the distances between thin filaments within the Z band showing transverse linking by presumed alpha -actinin filaments. The mean lattice spacing was calculated by dividing by 10 the distances covered by 11 filaments in 2 orthogonal rows like those indicated by the arrows. Clusters of putative glycogen granules (G) are present. Bar = 95 nm.

Thin filament densities were also measured as performed previously, using three standard grid squares positioned near the Z band in myofibrils that contained thin filaments in a square-array pattern (Fig. 2B) (22). To estimate the proportion of short thin filaments, i.e., the percent not reaching the A band-sampled region, filament concentrations were determined both in the I band within ~150 nm of thin filament origin at the Z band and where thin filaments first overlap with thick filaments in the A band (Fig. 2A). The proportion of short filaments was calculated by comparing the density of thin filaments in the overlap A-band region with that near the Z band (Fig. 2, A and B). This approach detects thin filaments too short to penetrate the overlap region of the A bands but does not determine absolute filament lengths. Whether a short filament, arising from the Z band, reaches the A band depends on sarcomere length. The present approach is valid because the variation in sarcomere lengths between the sampled fibers is small. Sarcomere length averages 2.5 ± 0.1 µm within each of the three gastrocnemius fiber types, and this length is not significantly different from that (2.3 ± 0.1 µm) of the soleus type I fibers. Furthermore, the within-subject pre- and postflight sarcomere lengths are similar because the mean ratio of pre- to postflight sarcomere lengths was near unity (0.96 ± 0.03).

Data presented in this paper show that Z bands expand at short sarcomere lengths in gastrocnemius fibers but not in soleus fibers (see Z-band lattice spacing and sarcomere length). This means that in the soleus, unlike gastrocnemius, at short sarcomere lengths thin filaments near the Z bands remain closely packed, whereas myofilaments in the overlap A-band region spread and decrease in density. To adjust for sarcomere length effects on density in soleus fibers, thin filament densities in the A bands were normalized to 2.3 µm (soleus mean length) before comparing the near Z-band and overlap A-band densities for short thin filaments. Additional confidence in the present approach for detecting short filament populations was gained by specifically comparing subsets of fibers in which sarcomere lengths of the pre- and postflight conditions were identical, and no normalization of sarcomere length was necessary. For the percentages of short filaments reported in this study, short filaments in the gastrocnemius are, on the average, <0.5 µm, and those in the soleus are <0.4 µm. Maximum thin filament length in humans is reportedly 1.5 µm (32). The present study does not investigate thin filament length variation >0.5 µm.

To determine whether the Z band lengthened in the transverse dimension at short sarcomere lengths, the lattice spacing of thin filaments interconnected by Z-band filaments within the Z band was measured and correlated with the sarcomere length for the same fiber. Mean spacing was determined in micrographs at ×201,000 of cross-sectioned fibers by dividing by 10 the distances spanned by two orthogonal rows of 11 nearest-neighbor thin filaments (Fig. 2B).

Physiological and biochemical measurements. On the day of testing, a muscle sample was transferred from skinning solution into cold (4°C) relaxing solution. As previously described, a 4- to 5-mm segment of a single muscle fiber was isolated and transferred to a small glass-bottomed chamber where the fiber ends were securely fastened to a force transducer (model 400, Cambridge Technology, Watertown MA) and motor (model 300B, Cambridge Technology) (34, 35). To ensure that the function of the sarcoplasmic reticulum was completely disrupted, the mounted fiber was briefly bathed in relaxing solution containing 0.5% Brij-58 (polyoxyethylene 20 cetyl ether, Sigma Chemical, St. Louis, MO).

The transducer, motor, and mounted fiber were placed on the stage of an inverted microscope. Sarcomere length was adjusted to 2.5 µm using a calibrated eyepiece micrometer. Fiber length, defined as the length of the fiber suspended between the force transducer and motor, was measured as the fiber was advanced across the magnified field of view by a digital micrometer. Analog outputs from the transducer and servomotor were monitored on a digital oscilloscope before being amplified, digitized, and interfaced to a personal computer. In-house software performed on-line analysis of the force and position data (34). Fibers were transferred from relaxing to activating (-log free Ca2+ concentration = 4.5) solution, allowed to attain peak force, and subjected to either rapid slack length steps (complete within 2 ms) and/or a series of isotonic load clamps. For the slack steps, the linear relationship between the time for tension redevelopment vs. slack step distance was used to determine fiber unloaded shortening velocity. All experiments were conducted at 15°C. Detailed descriptions and illustrations of these procedures have been presented in our laboratory's previous work (34, 35).

After the physiological measurements, the fiber segment was solubilized in 10 µl of an SDS sample buffer and stored at -80°C. Fibers were later run on 5% and 12% SDS-polyacrylamide gels and silver stained to identify fiber myosin heavy chain and myosin light chain isoform content, respectively.

Data were analyzed using ANOVA in which the spaceflight treatment (preflight, postflight) was nested within subjects (astronauts A, B, C, and D). If ANOVA revealed a significant treatment within subject effect, interpreted as an indication that fibers from at least one astronaut displayed a significant change with the spaceflight treatment, pre- vs. postspaceflight responses for each individual subject were evaluated with a t-test. For all tests, statistical significance was accepted at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Fiber types and thick filament densities. When normalized to 2.5-µm sarcomere length, the average thick filament densities in gastrocnemius fibers were not significantly different preflight between types I (1,015 ± 43 filaments/µm2), IIa (1,003 ± 33/µm2), and IIa/IIx (1,021 ± 36/µm2). Using these densities, mean thick filament spacing calculated for types I, IIa, and IIa/IIx was 31.4, 31.6, and 31.3 nm, respectively. After 17 days of spaceflight, the postflight thick filament densities remained similar between fiber types and unchanged (P = 0.2) from preflight values (type I 996 ± 55/µm2, IIa 1,004 ± 47/µm2, and IIa/IIx 1,018 ± 36/µm2). For these fibers, average thick filament spacing was 31.7, 31.6, and 31.3 nm, respectively. The thick filament density for soleus type I fibers normalized to 2.5 µm was similar preflight (1,028 ± 9 filaments/µm2) and postflight (1,012 ± 7/µm2). The calculated average filament spacings of 31.2 nm before and 31.4 nm after spaceflight for soleus were comparable to gastrocnemius. Whereas the packing densities of thick filaments were unchanged, the total amounts of thick filaments were presumably decreased postflight in astronauts B and D because fiber diameters were significantly decreased by 5.6 to 12.5% for gastrocnemius I and IIa fibers, whereas no atrophy was detected in IIa/IIx fibers of astronauts B and D or in any fiber type of astronauts A and C (35). The mean diameter of soleus type I fibers decreased by 8.3% for astronauts A, B, C, and D (34).

Thick filament spacing vs. sarcomere length was plotted for fiber types in the pre- and postflight biopsies of gastrocnemius and soleus. Filament spacing increased at shorter sarcomere lengths for all fiber types (Fig. 3). The slope of the curve for soleus type I fibers (m = -7.1) was steeper than those of the three gastrocnemius fiber types IIa/IIx, IIa, and I (m = -3.0, -2.5, and -2.3, respectively). This indicates greater spreading of thick filaments at short sarcomere lengths for soleus than gastrocnemius (Fig. 3).


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  		Fig. 3.   For type IIa/IIx, IIa, and I fibers, thick filament spacing in the A band is wider in fibers fixed at shorter sarcomere lengths. This is consistent with sarcomere transverse expansion during shortening. A: gastrocnemius type IIa/IIx fibers. B: gastrocnemius type IIa fibers. C: gastrocnemius type I fibers. D: soleus type I fibers.

Z-band lattice spacing and sarcomere length. For individual type I and IIa gastrocnemius fibers and type I soleus fibers, thin filament spacing (nearest neighbor) within the Z-band square-array lattice was measured and correlated with sarcomere length to ascertain whether the Z band expanded transversely at shorter sarcomere lengths. Spacing increased with declining sarcomere length for both gastrocnemius fiber types but not for soleus (Fig. 4).


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  		Fig. 4.   Type IIa (A) and I (B) fibers in pre- and postflight gastrocnemius muscles fixed at shorter sarcomere lengths show concomitant transverse expansion of the Z-band lattice spacing. Type I (C) fibers in pre- and postflight soleus muscles do not exhibit similar changes in lattice spacing, suggesting that soleus Z bands are more rigid than those in gastrocnemius fibers.

Thin filament density and length changes after spaceflight. The mean thin filament densities in the I band and in the overlap A band for type IIa/IIx and IIa fibers obtained preflight were not different from the postflight values (Table 1). In contrast, significant decreases in the average thin filament densities occurred postflight in gastrocnemius and soleus type I fibers (Table 1). In the preflight controls, the mean percentage of short thin filaments was 2.4-3.0 times higher in gastrocnemius type I, IIa, and IIa/IIx fibers compared with soleus type I fibers (Table 2). The percentages of short filaments were essentially unchanged after spaceflight for gastrocnemius type IIa/IIx and IIa fibers, but the type I fibers in both gastrocnemius and soleus had significantly higher occurrences of short filaments (Table 2).

                              
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  		Table 1.   Thin filament densities in astronaut muscles


                              
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  		Table 2.   Percentages of short thin filaments in astronaut muscles

Specific tension and thin filament density. In our companion paper on gastrocnemius physiology, the averaged specific tension for the four astronauts after spaceflight was not significantly altered for fiber types expressing type I, IIa, or IIa/IIx myosin heavy chains, although a small reduction in average absolute force was observed for the type I fibers (35). However, in the present study, plotting specific tension vs. thin filament density in the overlap A band for individual astronauts revealed that lower specific tension correlated directly with lower thin filament density in gastrocnemius type IIa/IIx, IIa, and I fibers (Fig. 5). Interestingly, soleus type I fibers, which had the highest amounts of thin filaments preflight, lacked a similar correlation (Fig. 5).


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  		Fig. 5.   Average specific tension decreases as thin filament densities decrease in the overlap A band regions of pre- and postflight gastrocnemius IIa/IIx (A), IIa (B), and I (C) fiber types. The type I (D) fibers of the soleus do not show a fall in specific tension at lower thin filament density levels. The lack of a decrease in soleus fibers may reflect the higher thin filament densities preflight and the slow myosin in this fiber type.

Shortening velocity and thin filament density. Thin filament density in the overlap A band was inversely related (R = 0.98) to velocity of shortening for the pooled soleus type I fibers from astronauts A, B, C, and D (Fig. 6). For gastrocnemius, the velocity vs. density data were plotted separately for astronaut pairs AD and BC because their control preflight densities of thin filaments in the overlap A bands were consistently higher in both astronauts A and D compared with astronauts B and C for IIa/IIx (2,956 ± 125 vs. 2,554 ± 102 µm2), IIa (2,580 ± 1 vs. 2,241 ± 170 µm2), and I (2,065 ± 33 vs. 1,577 ± 21 µm2) fibers (Table 1). Similar differences did not exist for soleus type I fibers (astronaut pair AD = 2,320 ± 186 vs. astronaut pair BC = 2,297 ± 190 µm2). In all cases, the postflight values were lower for thin filament density and higher for velocity of shortening compared with preflight (Fig. 6). Velocity and thin filament densities were also inversely correlated for gastrocnemius type I fibers (R = 0.79 for astronaut pair AD and 0.90 for astronaut pair BC) and type IIa fibers (R = 0.97 for astronaut pair AD and R = 0.66 for astronaut pair BC) (Fig. 6). The increase in velocity was steeper for gastrocnemius type I fibers of astronaut pair BC with lower preflight densities than astronaut pair AD (Fig. 6).


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  		Fig. 6.   For soleus (Sol) type I fibers in the 4 astronauts (A, B, C, and D), thin filament densities are higher preflight than postflight. Velocity of shortening is higher for lower thin filament density. Astronaut pair AD has higher pre- and postflight thin filament densities than astronaut pair BC. For gastrocnemius (Gastroc) type IIa and I fibers, the rightmost point for each curve represents preflight values, and the leftmost point is postflight. FL, fiber length.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Thick and thin filament properties. Across the fast and slow fiber types in gastrocnemius and soleus muscles of the astronauts, thick filament densities standardized to a sarcomere length of 2.5 µm were essentially the same both before and after spaceflight. This indicates that muscle cells maintain thick filaments at a relatively constant packing density of 1,012 ± 5 filaments/µm2 or 31-nm mean spacing distance independent of the myosin isoforms present in the sarcomere. In striking contrast, thin filament density and length vary markedly across fiber types in normal fibers and change after unloading. Consistency of thick filament lengths and variability of thin filament lengths occurs within different species for both skeletal and cardiac muscle (9, 10, 15, 24, 31, 32). This suggests that thin filament diversity is an important factor contributing to the functional adaptability of muscle.

In preflight muscles, slow and fast fibers of the gastrocnemius possess a higher percentage (~23%) of short thin filaments than that in soleus slow fibers (9%). This means that the occurrence of short thin filaments is muscle-type specific rather than fiber-type specific because the slow type I fibers in gastrocnemius have features in common with the fast fibers in gastrocnemius rather than in common with soleus slow type I fibers. On the other hand, after spaceflight, the slow soleus fibers acquire greater percentages (increasing from 9 to 24%) of short thin filaments comparable to gastrocnemius fiber types. Short filament content in gastrocnemius type I fibers is also higher (rising from 26 to 31%) postflight. This indicates that slow fibers adapting to spaceflight unloading acquire the fast muscle characteristic of more short filaments.

The higher percentage of short filaments in fast muscles may reflect greater dynamic instability of all of the actin thin filaments, i.e., shrinkage and regrowth occurring more frequently than in slow muscles. Thin filament instability may be linked to muscle use because actin gene expression can be modulated by mechanical forces (17). Short and long thin filaments may represent distinct functional populations. There is growing evidence for regulation of filament length by nebulin isoforms, which generate short and long filaments (15). Reduced loading of soleus is expected to shift from a tonic to a phasic (fast muscle) pattern of contractile activity (11). Altered activity can modulate muscle protein isoform expression, although nebulin has not been specifically examined (11, 16). In cardiac muscle cells, nebulin-related nebulette isoforms appear to be responsible for the heterogeneity of thin filament lengths (20). Thin filaments are stabilized by tropomyosin isoforms that interact with tropomodulins and CapZ capping proteins, and the tropomodulins exhibit fast and slow muscle isoform diversity (18). Transfection overexpression of tropomodulin results in abnormally short thin filaments (18, 27). Thus there exists a large repertoire of thin filament-associated proteins in which the richness of isoform type and amount could modulate thin filament structure and muscle function.

Sarcomere architecture at different sarcomere lengths. When muscle fibers are aldehyde fixed at different sarcomere lengths as in the present study, the distances between thick filaments and between thin filaments are greater at shorter lengths for all fiber types in gastrocnemius and soleus. X-ray diffraction of living fibers and electron microscopy studies over many years have demonstrated that transverse expansion of the myofilament lattice with sarcomere shortening is a universal principle across species for skeletal and cardiac muscle (8, 12, 23). For the astronauts, the expansion is similar in magnitude for gastrocnemius I, IIa, and IIa/IIx fiber types but more than twofold greater for soleus type I fibers.

Our measurements indicate that, as thick and thin filaments move further apart transversely at shorter sarcomere lengths, the Z bands also expand transversely in gastrocnemius fibers but not soleus fibers. Z-band expansion is consistent with reports for slow and fast fibers fixed at different lengths in noncontracting relaxed muscles (3, 8). However, Z bands are expanded in fibers fixed when actively contracting (8). The lack of Z-band expansion in fixed-relaxed soleus fibers most likely reflects the greater structural protein content and stiffness of their wide Z bands compared with the more extensible narrow Z bands in fast fibers (37). Narrow Z bands apparently offer less resistance to stretching during passive shortening than wide Z bands. The higher tension of active contraction is needed to stretch the series elastic Z-band lattice elements (8). Z-band stiffness is not simply related to width because type I fibers in gastrocnemius and soleus both have wide Z lines, but the gastrocnemius Z bands exhibit greater extensibility. Z bands contain isoforms of many known proteins, and uncharacterized proteins exist, which collectively determine rigidity (28).

Specific tension, shortening velocity, and thin filament density. Variations in thin filament density and length appear to affect contractile properties. Specific tension correlates directly with thin filament packing density in the three gastrocnemius fiber types. Two of the four astronauts had higher thin filament densities before flight, and their fibers exhibited higher specific tensions. This suggests that muscle strength (force per myofibril cross section) may be modulated by thin filament packing density. It is unclear whether the density differences present in the two pairs of astronauts before flight were due to exercise lifestyle or genetic variation. A larger number of subjects is required to explore this issue. For soleus, specific tension did not correlate with thin filament density. Soleus fibers had the highest thin filament densities preflight, and loss of thin filaments may not have impacted specific tension postflight because sufficient numbers of thin filaments remained in the slow muscle to sustain the required levels of cross-bridge formation.

Many studies have reported that, during the early phases of exercise strength training, muscle strength can increase dramatically without measurable muscle hypertrophy. This phenomenon has generally been attributed to enhanced efficiency of motor unit recruitment, decreased activity of antagonist muscles, increased angle of fiber attachment, and decreased connective tissue per cross-sectional area (4, 13, 21). Our finding that specific tension and thin filament density in single muscle fibers are directly correlated in gastrocnemius fiber types opens the possibility that changing the packing density of thin filaments within sarcomeres is a mechanism that alters fiber strength before fiber hypertrophy and atrophy are detectable by techniques such as magnetic resonance imaging and light microscopic histology. Interestingly, the four astronauts in the present study show no significant changes in calf muscle strength tested in vivo (30). This indicates that the single-fiber structural and physiological alterations precede whole muscle changes, although greater sensitivity of single-cell measurements is the most likely explanation. Until temporal studies of specific tension and thin filament packing density in single fibers are completed on multiple biopsies during strength training and during unloading deconditioning, the correlation between packing density and strength cannot be distinguished as a causative rather than a secondary response to altered mechanical stimuli.

Higher thin filament density is associated with lower velocity of shortening in all fiber types of the astronauts. This relationship was observed previously for bed-rest subjects (22, 33). Soleus fibers show reduced thin filament densities within the overlap region of the A band during spaceflight and bed-rest unloading, and there are marked increases in the velocity of shortening of the slow fibers in the absence of a shift from slow to fast myosin (33-35). Thin filament density may also modulate velocity of shortening in smooth muscle, indicating that this is a general principle for muscle tissue (19). A high density of thin filaments appears important for muscles that perform slow tonic activities, such as postural maintenance and sphincter action.

Summary. In astronaut fast and slow skeletal muscles before and after spaceflight, thick filament packing density is remarkably uniform across muscles and muscle fiber types, whereas thin filaments can vary significantly in packing density and length. Thin filament variability correlates with functional diversity and may confer muscle fiber adaptability. Temporal studies are necessary to explicitly define cause and effect in these structure-function relationships. Although major changes in muscle strength depend on muscle fiber area, modulating thin filament properties may play a role in altering specific tension (strength) and shortening velocity at the subcellular level in muscle fibers adapting to the diverse environments of Earth and space.


    ACKNOWLEDGEMENTS

This research was supported by National Aeronautics and Space Administration Grants NAS9-18768 (to R. H. Fitts) and NAG2-956 (to D. A. Riley) and National Institute of Neurological Disorders and Stroke Grant U01 NS-33472 (to D. A. Riley).


    FOOTNOTES

Address for reprint requests and other correspondence: D. A. Riley, Dept. of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI (E-mail: dariley{at}mcw.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/japplphysiol.00717.2001

Received 10 July 2001; accepted in final form 10 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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