Cross-sectional area (CSA), peak Ca2+-activated force (Po), fiber specific force (Po/CSA), and unloaded shortening velocity (Vo) were measured in slow-twitch [containing type I myosin heavy chain (MHC)] and fast-twitch (containing type II MHC) chemically skinned soleus muscle fiber segments obtained from three strains of weight-bearing and 7-day hindlimb-suspended (HS) mice. HS reduced soleus slow MHC content (from ∼50 to ∼33%) in CBA/J and ICR strains without affecting slow MHC content in C57BL/6 mice (∼20% of total MHC). Two-way ANOVA revealed HS-induced reductions in CSA, Po, and Po/CSA of slow and fast fibers from all strains. Fiber Vo was elevated post-HS, but not consistently across strains. No MHC × HS treatment interactions were observed for any variable for C57BL/6 and CBA/J mice, and the two significant interactions found for the ICR strain (CSA, Po) appeared related to inherent pre-HS differences in slow vs. fast fiber CSA. In the mouse HS models studied here, fiber atrophy and contractile dysfunction were partially dependent on animal strain and generally independent of fiber MHC isoform content.
- muscle atrophy
- hindlimb unweighting
- hindlimb unloading
- non-weight bearing
hindlimb suspension (HS) is the one of the most commonly used animal models of musculoskeletal non-weight bearing. The model was originally developed as a ground-based procedure to mimic the effects of spaceflight on laboratory rats (29). Soon after its introduction, the model was successfully adapted for mice (37) and later for other laboratory species, such as hamsters (6) and rabbits (2). Despite the successful application of the HS model to a number of common laboratory mammals, studies using rats have dominated the HS literature (for review see Refs. 15, 38).
The soleus is particularly susceptible to HS-induced atrophy. In the rat, the soleus is a classic slow-twitch muscle in which ≥85% of the fibers express the type I myosin heavy chain (MHC) isoform (4, 12) or are classified as type I by myosin ATPase histochemistry (8). Studies conducted on permeabilized or skinned single-fiber segments have shown that, in addition to gross fiber atrophy, cellular mechanisms underlying force production and sarcomere shortening are altered after HS in those fibers that contain type I MHC (16, 31). One of the strengths of this single-fiber approach is that changes in cell function can be related to fiber protein isoform content. However, because of the fiber-type composition of the rat soleus, physiological data on fast-twitch fibers from this model are very limited, making comparisons between the responses of slow and fast fibers difficult. For instance, in a recent study, only 3 and 8 fast soleus fibers were isolated from weight-bearing (WB) and 15-day HS rats, respectively, vs. the 26 and 22 slow fibers that were isolated from the same muscles (32). Such low numbers of fast fibers make statistical analysis and interpretation difficult.
To evaluate the susceptibility of slow vs. fast fibers to HS, single cells have been isolated from hindlimb muscles containing substantial populations of fast fibers, such as the gastrocnemius (16) or plantaris (31). Fast fibers isolated from these muscles typically show fewer functional changes than slow soleus fibers. One must be aware that comparisons of this type are conducted between fibers differing not only in their MHC isoform content but also in their muscle of origin. The latter may be an important consideration because hindlimb muscles experience substantially different recruitment and loading patterns during normal WB activity (39) and thus potentially large differences in the relative change in mechanical load during HS. An alternative experimental approach that avoids this issue involves comparing slow and fast muscle fibers obtained from a single hindlimb muscle of mixed fiber-type composition, such as the rat gastrocnemius (16). The limitation of this approach is that, during HS, the rat gastrocnemius atrophies to a lesser extent than the soleus (30).
In contrast to the rat, the mouse soleus is composed of substantial populations of both slow and fast fibers, with the fast fiber population accounting for ∼25–65% of total fibers, or total MHC, depending on mouse strain (5, 19, 25, 40, 41). Thus the mouse soleus, with an inherent susceptible to atrophy combined with a mixed fiber-type composition, would be an ideal muscle to test the susceptibility of slow and fast fibers to HS-induced atrophy and dysfunction. In the present study, a mouse model was used to evaluate the functional responses of slow vs. fast muscle fibers to 7 days of HS. Our results show that for three mouse strains, slow and fast soleus fibers undergo functional adaptations during HS and, with some exceptions between strains, the magnitude of these changes are generally similar across fiber type.
Animal care and suspension procedure. All procedures involving animals were approved by the Oregon State University Institutional Animal Care and Use Committee. Initially, we chose to study C57BL/6 mice because of the wide use of this strain in biomedical research (13). As will be discussed, the results of this initial study prompted us to perform two additional studies in which we examined the responses of ICR (or CD-1) and CBA/J strains to the same experimental treatments.
The C57BL/6 mice were obtained from Simonsen Laboratories (Gilroy, CA). The ICR and CBA/J mice were obtained from Harlan Laboratories (Indianapolis, IN). All mice were males between 8 and 12 wk of age. On arrival, mice were housed at Oregon State University's Animal Care Facility for a minimum of 7 days. Animals were then randomly assigned to a HS or a WB group.
The mice in the HS group were suspended for 7 days by a tail harness. The HS procedure was modified from the rat HS method described by Fitts et al. (14). After the tail had been cleaned, a base layer of adhesive tape was secured to the proximal two-thirds of the tail by cyanoacrylate glue. A piece of 18-gauge wire, shaped to form a triangle, was attached to this base layer with additional strips of adhesive tape. A fishing swivel was clipped into the wire triangle. Monofilament line tied to the swivel was used to raise the animals' hindlimbs off a grid floor that made up the base of the HS apparatus. The monofilament line passed through a small hole in a sheet of Plexiglas that formed the top of the suspension apparatus. The length of the line was adjusted to prevent the animals hindlimbs from touching the grid floor or other supportive surfaces. The forelimbs maintained contact with the grid floor, allowing the animal to move about in a circular area to gain access to food and water. Waste passed through the grid floor onto absorbent paper. The exposed tip of the tail was monitored daily to ensure adequate blood flow.
The WB animals were housed in standard cages (1 mouse per cage) in the same room as the HS animals. This room was maintained at 23°C with a 12:12-h light-dark cycle. All WB and HS groups were fed a similar ad libitum diet of laboratory chow and tap water.
Tissue dissection and preparation. Mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (40 mg/kg body mass). The HS mice were anesthetized while still suspended to prevent any reloading-induced muscle damage. The left soleus was excised, trimmed of tendon, weighted, and immediately placed in cold (∼4°C) dissection solution (for composition, see Solutions). The right hindlimb muscles, including the soleus, plantaris, gastrocnemius, tibialis anterior (TA), and extensor digitorum longus (EDL), were removed, trimmed of connective tissue and fat, and weighed. The right soleus muscles were then frozen in liquid nitrogen and stored at –80°C.
The left soleus muscle was divided longitudinally into two or three bundles. The muscle bundles were transferred into skinning solution (for composition, see Solutions) and stored at 4°C. After 24 h at 4°C, the fiber bundles were transferred into fresh skinning solution and stored at –20°C for up to 4 wk.
Solutions. The computer program and stability constants (adjusted for pH, temperature, and ionic strength) described by Fabiato (10, 11) were used to determine the composition of the relaxing and activating solutions used for the physiological experiments. Relaxing and activating solutions contained 7.0 mM EGTA, 20.0 mM imidazole, 4 mM Mg2+-ATP, 1 mM free Mg2+, 14.5 mM creatine phosphate, and 15 U/ml creatine kinase. A CaCl2 standard solution (calcium molarity standard, Corning, Corning, NY) was used to adjust the free Ca2+ concentration of the relaxing and activating solutions to pCa 9.0 and pCa 4.5, respectively (where pCa = –log Ca2+ concentration). The pH of both solutions was adjusted to 7.0 with KOH and the total ionic strength to 180 mM with KCl. The dissection solution was composed of protease inhibitors dissolved in relaxing solution according to the directions of the manufacturer (Complete Mini EDTA-Free Protease Inhibitor Tablets, Boehringer Mannheim, Indianapolis, IN). The skinning solution was composed of equal volumes of dissection solution and glycerol. Protease inhibitors were not present in the experimental relaxing or activating solutions.
Single-fiber functional experiments. Single-fiber segments were isolated from muscle bundles by using fine forceps. The ends of an isolated fiber segment were mounted between an isometric force transducer (model 400, Aurora Scientific, Aurora, Ontario, Canada) and a direct-current position motor (model 308B, Aurora Scientific) as previously described (42). The mounted fiber was suspended in one of several small wells milled into a stainless steel dip plate, allowing rapid transfer of the fiber from well to well. A small thermocouple was used to monitor solution temperatures during relaxation and activation. Solution temperatures were maintained at 15°C throughout all experiments.
The dip plate was mounted on the stage of an inverted microscope (Olympus IX-70, Olympus America, Melville, NY). Sarcomere length was adjusted to 2.5 μm with the aid of an ocular micrometer. Fiber length (FL) was determined by using a digital micrometer that translated the microscope stage. Fiber cross-sectional area (CSA) was calculated by measuring the diameter of the fiber while it was briefly (<5 s) suspended in air. Fiber CSA was calculated from the width measurement assuming that the fiber forms a cylinder in air (28). Three separate measurements were conducted along the length of the fiber (with the fiber returned to relaxing solution between measurements), and the final fiber CSA was taken as the mean of the three CSA values.
Output from the motor and transducer were monitored on a digital oscilloscope (model Integra 10, Nicolet Technologies, Madison, WI) and amplified by a differential amplifier (model DIF2, Positron Development, Inglewood, CA). The amplified signals were digitized (5 kHz) and interfaced to a personal computer by a data-acquisition board (model AT-MIO-16E, National Instruments, Austin, TX). Data acquisition, storage, and analysis were controlled through custom programs written in our laboratory (LabView, National Instruments).
Unloaded shortening velocity (Vo) and peak Ca2+-activated force (Po) were determined by the slack test method (Fig. 1). All slack steps were ≤20% of FL. Absolute Po was determined as the difference between the maximum force achieved before the fiber was slacked and the baseline force acquired during the period of unloaded shortening. Fiber specific force (Po/CSA) was calculated by dividing Po by fiber CSA. At the conclusion of the slack test, the fiber was removed from the apparatus, placed in 30 μl of SDS sample buffer, denatured at 95°C for 4 min, and stored at –80°C.
Gel electrophoresis. Myosin was extracted from the whole muscle samples (23). As previously described (42), a discontinuous polyacrylamide gel system was used to separate MHC isoforms in whole muscle extracts and in single skinned fiber segments, with the protein bands subsequently visualized by silver staining. Relative MHC isoform composition of whole muscle myosin extracts were assessed with laser-scanning densitometry (ImageQuanT 5.0, Molecular Dynamics, Sunnyvale, CA). Gels were scanned for 100-μm pixel size, and area integration was performed to determine relative density of each MHC band present.
Statistical analysis. Data are presented as means ± SE. For each study, WB vs. HS differences in soleus muscle mass and MHC isoform content were evaluated with a t-test. Fiber CSA, Po, Po/CSA, and Vo were evaluated by a two-way ANOVA (main effects of MHC isoform content and HS treatment). For the two-way analysis, Tukey's post hoc procedure was used to evaluate 1) significant main effects consisting of more than two levels and 2) significant interactions between MHC isoform content and HS treatment. Statistical significance was accepted at P < 0.05.
Study 1: Response of C57BL/6 mice to 7 days of HS. Soleus mass decreased 24% after 7 days of HS (Table 1). HS also reduced the mass of the plantaris (–17%), gastrocnemius (–22%), and TA (–11%), but it did not affect the mass of the EDL.
Figure 2A is a silver-stained SDS gel showing single muscle fiber segments expressing or coexpressing the four MHC isoforms present in adult mouse skeletal muscles. Because the IIx isoform migrated to a position aligning with the very bottom of the type IIa band, it was difficult to determine whether a fiber containing the IIa isoform expressed this isoform exclusively or whether it also coexpressed the IIx MHC. This migration pattern also made it difficult to interpret the slowest migrating band on soleus homogenate gels (Fig. 2B). Consistent separation of the mouse IIa and IIx isoforms seems to be more difficult than separation of the rat IIa and IIx MHCs because we were able to clearly separate these latter two isoforms by using the same gel system (42). Similar difficulty in the electrophoretic identification of the IIx isoform in skeletal muscle homogenates of mice has been reported by several other research groups (1, 25, 44). Because of the uncertainty regarding the exact composition of the slowest migrating band on our gels, we have designated this band IIa(x) to signify that the band, although clearly containing the type IIa isoform, may also contain some IIx MHC.
The soleus of WB animals (n = 7) was composed of 20.2 ± 2.5% type I MHC, 77.6 ± 2.9% type IIa(x) MHC, and 2.2 ± 2.2% type IIb MHC. Seven days of HS had no effect on the MHC isoform content of soleus muscles obtained from the C57BL/6 mice [n = 7; 21.6 ± 2.1% type I; 75.5 ± 3.4% type IIa(x); 2.9 ± 2.0% type IIb; P > 0.05 for all comparisons]. The relatively low expression of the type I MHC isoform was somewhat unexpected on the basis of previous data for this strain (25). Therefore, to verify our whole muscle MHC extract results, skinned fibers were isolated from soleus muscle bundles and subjected to the same electrophoretic procedure as the whole muscle MHC extracts. In single fibers obtained from 10 WB mice (an average of 41 fibers per mouse), type I, IIa(x), and IIb fibers accounted for 18.9 ± 2.1, 66.4 ± 3.1, and 1.3 ± 10.9% of the total fibers, respectively, with 5.0 ± 1.1, 2.0 ± 1.3, and 6.3 ± 2.2% of the fibers coexpressing I with IIa(x) MHC, IIa(x) with IIb MHC, and IIx with IIb MHC, respectively. Single fibers obtained from 9 HS mice (an average of 48 fibers per mouse) showed a similar distribution, with 22.3 ± 2.8 and 64.0 ± 3.0% of the fibers containing type I or IIa(x) MHC (no fibers were detected that contained type IIb MHC exclusively), and 5.2 ± 1.3, 4.1 ± 1.1, and 4.5 ± 2.4% of the fibers containing type I/IIa(x), type IIa(x)/IIb, and IIx/IIb isoforms (P > 0.05 for all comparisons to the WB samples).
The effect of 7 days of HS on the CSA, Po, Po/CSA, and Vo of fibers expressing slow or fast MHC isoforms are compiled in Fig. 3. Fast fibers were subclassified as those expressing the IIa(x) MHC and those coexpressing the type IIx and IIb MHCs. Two-way ANOVA revealed a significant main effect of fiber MHC isoform content across all four variables. Tukey's post hoc procedure indicated that fiber CSA and absolute Po followed a pattern in which type IIx/IIb fibers > type I fibers > type IIa fibers, with each group significantly different from the others. Type IIx/IIb fibers produced greater Po/CSA than type IIa(x) fibers, but the magnitude of this difference was only 6%. As expected, Vo was fastest in type IIx/IIb fibers, intermediate in type IIa(x) fibers, and slowest in fibers expressing type I MHC (all comparisons P < 0.05).
A significant main effect of HS treatment was noted for all variables except Vo. Seven days of HS reduced fiber CSA by 33, 33, and 16%, fiber Po by 46, 47, and 33%, and fiber Po/CSA by 15, 20, and 17%, in fibers expressing type I, IIa(x), and IIx/IIb MHC, respectively. The absence of any significant MHC × HS treatment interactions indicated that the HS-induced changes in fiber CSA, Po, and Po/CSA were similar for all three groups of fibers.
Fiber Vo is an inverse log function of species body mass (36). Compared with previous work conducted in our laboratory on rat fibers (43), the Vo of the type I fibers obtained from the C57BL/6 mice seemed relatively slow. To examine this further, we used published allometric relationships (36) to predict the Vo (at 15°C) for type I fibers obtained from our mice. Figure 3 shows that the average Vo of the type I fibers obtained from the C57BL/6 mice, 1.52 ± 0.08 FL/s, is substantially less than the predicted value of 1.93 FL/s. To check whether this discrepancy could be related to differences in methodology between laboratories (e.g., the exact chemical composition, pH, and ionic strength of the activating solutions), we also measured the Vo of type IIb fibers obtained from the gastrocnemius of these same C57BL/6 mice. As shown in Fig. 4, the observed average Vo of 78 type IIb gastrocnemius fibers, 6.52 ± 0.17 FL/s, was almost identical to the value of 6.58 FL/s predicted for a species of this mass (36). Thus the lower than predicted Vo of the type I fibers from the C57BL/6 mice could not be explained by differences in experimental conditions between the present investigation and those studies used to develop the allometric relationships.
The lower than predicted Vo of the type I fibers, plus the relatively low type I MHC composition of the soleus, caused us to question whether the C57BL/6 mice were representative of other mouse strains. This prompted us to perform additional studies using other strains of mice.
Study 2: Response of ICR mice to 7 days of HS. The ICR mouse stain was chosen for this study because of its use in previous HS studies (5, 21, 40) and because soleus muscles from this strain have been reported to contain >50% type I MHC (5, 40). Seven days of HS reduced the mass of the ICR soleus by 43%, the plantaris by 21% (P = 0.06), the gastrocnemius by 27%, the TA by 23%, and the EDL by 20% (Table 1). No type IIb MHC was observed in the soleus muscle MHC extracts obtained from either WB or HS ICR mice. In WB ICR mice, slow MHC accounted for 49.2 ± 2.4% (n = 6) of total MHC. This differed significantly from the HS ICR mice where slow MHC made up only 31.8 ± 3.3% (n = 4) of total MHC (P < 0.01). In both groups of mice, the remaining MHC was classified as type IIa(x).
None of the single fibers isolated from the soleus of this strain contained type IIb MHC. The effect of HS on the CSA and functional properties of fibers expressing type I or type IIa(x) MHC are compiled in Fig. 5. As expected, type I fibers had a significantly slower Vo than type II fibers. Type I fibers were larger in CSA and produced greater absolute Po than the type II fibers. Po/CSA did not differ between slow and fast fibers.
Seven days of HS caused significant reductions in fiber CSA, Po, and Po/CSA, along with significant increases in the Vo of the ICR soleus fibers. Changes in Po/CSA (–13% for both type I and IIa fibers) and Vo (+14 and +12% for type I and II fibers, respectively) were independent of fiber type. However, significant MHC × HS treatment interactions revealed fiber typespecific changes in CSA and absolute Po with HS. Thus the relative reductions in CSA and Po for the type I fibers (39 and 47%, respectively) were greater than the reductions observed for the type IIa(x) fibers (21 and 31%, respectively). Further analysis of these interactive effects by Tukey's post hoc procedure indicated that the fast fibers were significantly smaller in CSA and produced less Po than slow fibers under WB conditions but that they were similar in CSA and produced the same Po as slow fibers after HS.
Study 3: Response of CBA/J mice to 7 days of HS. For this study, we examined CBA/J mice because this strain has been reported to have a relatively high percentage of slow soleus fibers as determined by ATPase histochemistry (41). Seven days of HS reduced the mass of the CBA/J soleus by 22% (Table 1). Also displaying atrophy were the plantaris (–16%), the gastrocnemius (–15%), and the TA (–12%) but not the EDL. Slow MHC accounted for 49.0 ± 0.6% (n = 6) of total soleus MHC in WB CBA/J mice but only 34.6 ± 2.9% (n = 4) of total MHC in the HS animals (P < 0.001). In both strains, the remaining MHC was identified as type IIa(x).
All fibers isolated from the CBA/J soleus contained either type I or type IIa(x) MHC. As summarized in Fig. 6, the Vo of the type II fibers was significantly greater than that of the type I fibers. The slow fibers, regardless of HS treatment, also produced greater Po/CSA than the fast fibers, but the magnitude of this difference was quite small (<4%).
Slow and fast fibers showed significant reductions in fiber CSA (34 and 36%, respectively), Po (41 and 43%, respectively), and Po/CSA (11 and 12%, respectively) with no overall change in Vo. The absence of MHC × treatment interactions indicated that these changes in CSA, Po, and Po/CSA were similar for the slow and fast fibers.
The mammalian soleus is generally described as a slow muscle. Although this seems to hold true for several species, such as rats (4, 8, 12) and humans (22), it does not describe the soleus muscles of all mammals. In hamsters (6) and Thoroughbred horses (36), slow fibers account for only 55 and 23% of the total soleus fibers, respectively. Furthermore, in most laboratory mice, the soleus is a mixed muscle, showing wide interstrain variation in its fiber type composition (5, 19, 25, 40, 41). In this study, we took advantage of the mixed fiber type composition of the mouse soleus to assess the susceptibility of slow and fast soleus muscle fibers to HS-induced atrophy and contractile dysfunction. The present data are novel in that they allow a comparison to be made between slow and fast fibers obtained from the hindlimb muscle most susceptible to prolonged HS.
Soleus atrophy after HS. In agreement with previous studies using mice (5, 7, 19–21, 37, 40), HS caused significant reductions in the mass of the soleus, plantaris, gastrocnemius, and TA. Consistent with several of these studies, we also found that the extensor digitorum longus did not atrophy in C57BL/6 and CBA/J strains, although we did observe atrophy of this muscle in the ICR strain. The 24–43% reductions in soleus mass noted here are similar to 27–40% losses in soleus mass reported for various strains of mice in previous 7-day HS studies (5, 21, 37). Clearly, there is variability in the response of mice to HS but similar variability has been observed in 7-day rat HS studies, where losses in soleus mass have been reported to range from 20% (18) up to 40% (14, 27, 30). Therefore, as a first approximation, our data support the conclusion reached by Ingalls et al. (21) that the magnitude of soleus atrophy during HS appears similar for rats and mice.
HS-induced changes in fiber CSA and Po. Fiber diameter swells by 20% as a result of the chemical skinning procedure used in this study (17). Therefore, to compare our fiber CSA data with literature values, we adjusted the observed mean CSA values presented in the figures by a factor of 1.44 to estimate the CSA of the mouse soleus fibers before skinning. For the 8- to 12-wk-old animals used in this study, estimated pre-skinning mean CSA ranged from 986 (CBA strain) to 1,055 (C57BL/6) μm2 for fibers expressing type I MHC, from 728 (ICR) to 981 (CBA/J) μm2 for fibers containing type IIa(x) MHC, and 1,488 μm2 (C57BL/6) for the IIx/IIb fibers. Our adjusted values are in good agreement with histochemical CSA data obtained on 6- and 14-wk-old 129B6F1/6 mice: 1,103 and 941 μm2 for type I fibers and 804 and 931 μm2 for type IIa fibers (19).
We found significant HS-induced reductions in the CSA of both slow and fast soleus fibers, ranging from 33 to 39% in type I fibers, from 21 to 35% in type IIa fibers, and 16% in type IIx/IIb fibers. There are few data available on the response of mouse soleus muscle fibers to 7 days of HS, although Haida et al. (19) reported atrophy of 37–59% for type I fibers and 46–61% for IIa fibers after a twofold longer duration of HS (14 days).
Associated with fiber atrophy was a 31–47% reduction in fiber Po. Significant post-HS reductions in the specific force of slow and fast fibers indicated that absolute Po declined out of proportion to fiber atrophy. Our data indicate that this loss in the intrinsic ability of fibers to produce force was responsible for approximately one-third of the HS-induced loss in the absolute force of the fibers. In practice, these findings mean that relying on HS-induced reductions in mouse fiber CSA as an index of changes in fiber function will result in a substantial overestimation of the actual potential of atrophied slow and fast fibers to produce force.
The mechanisms responsible for this decline in fiber-specific force cannot be due to excitation-contraction failure because the skinned fibers were activated with a saturating concentration of Ca2+. The decrements in specific force after HS may be a mechanical manifestation of ultrastructural changes occurring with muscle atrophy, such as a loss of thin or thick myofilament density within myofibrils or a reduction in the density of myofibrils within a fiber (35). Both of these changes would be expected to reduce the number of potential cross bridges per unit CSA of fiber. Alternatively, HS could induce an alteration in thin filament regulatory proteins that prevents full activation of the thin filament, even in the presence of saturating free Ca2+, or a physical change to myosin S1 or the thin filament that reduces the force produced by each cross bridge. Additional work is necessary to sort out these possibilities.
We are unaware of any previous data describing the functional adaptations of mouse fibers to HS. However, Ingalls et al. (21) noted a 38% reduction in the peak tetanic force of the mouse soleus (ICR strain) after 7 days of HS. This reduction in tetanic force exceeded the reported loss in soleus mass by 29%, suggestive of a loss in muscle specific force. In fact, in another study from the same group (20), soleus specific force fell by 24% after 14 days of HS. On the basis of the agreement between the present single-fiber data and these whole muscle studies, it seems reasonable to assume that at least a portion of the soleus force deficit observed in the HS mouse model is attributable to the absolute and specific force deficits of individual muscle cells reported here.
Although there are insufficient fast fibers in the rat soleus to permit a valid comparison, it is possible to compare the response of slow soleus fibers obtained from rats and mice. After 7 days of HS, McDonald and Fitts (27) reported a 16% reduction in fiber width, equivalent to a 30% reduction in CSA, a 42% loss in Po, and a 18% drop in the Po/CSA of rat slow soleus fibers. These data are very similar to the 33–39% reduction in CSA, 41–47% reductions in Po, and 11–15% loss in Po/CSA observed for slow fibers obtained from the three mouse strains of the present study. On the basis of this comparison, we conclude that relative slow soleus fiber atrophy and associated changes in Po and Po/CSA are similar in rats and mice after 7 days of HS.
Atrophy and reductions in force of slow vs. fast fibers. With the exception of the ICR strain, we observed no significant interaction between fiber MHC isoform content and HS for fiber CSA, Po, or Po/CSA. Thus, in C57BL/6 and CBA/J mice, slow and fast fibers responded similarly to the 7-day HS procedure. As outlined in the introduction, the results of previous single-fiber studies conducted on rats suggest that slow fibers are preferentially affected by HS. The present data are not consistent with this interpretation. However, a relatively straightforward explanation of the present C57BL/6 and CBA/J data, in a manner that is consistent with previous rat soleus HS results, is possible if one considers the unique functional properties of the soleus. In the cat, the soleus is maximally or near maximally recruited during normal WB activity (39). If a similar recruitment pattern in the rat and mouse is assumed, the absence of WB activity would be expected to result in a similar relative reduction in mechanical load across all soleus fibers (or all but the very fastest) in both species. Because the absence of tonic mechanical loading may be a primary factor responsible for muscle fiber atrophy (35), one would predict that the signal for atrophy is experienced similarly by both slow and fast fibers in rats and mice.
The present C57BL/6 and CBA/J data are consistent with a model in which slow and fast soleus fibers display the same susceptibility to atrophy and dysfunction in response to the same relative reduction in mechanical loading. This interpretation, which is based on motor unit recruitment rather than fiber type or fiber MHC isoform content, is also consistent with previous rat HS results. In the rat, slow vs. fast fiber comparisons must out of necessity be conducted on fibers from different muscles, e.g.. the gastrocnemius. Unlike the soleus, motor units are recruited in this muscle in proportion to increasing intensity of locomotor activity (39). Because slow fibers in these muscles are more likely to be recruited to a greater extent during normal activities, they experience a greater relative reduction in mechanical load, and consequently greater atrophy, when the animal is subjected to HS.
The ICR mice do not appear to fit this model because fast fibers from this strain showed only one-half as much relative atrophy as the type I fibers. A unique characteristic of this strain was that the fast soleus fibers were 30% smaller in CSA than the slow fibers before HS. This contrasts to the WB CBA/J and C57BL/6 mice, in which the CSAs of the type I and type IIa(x) fibers were within 10%. Thus the initial or WB fiber CSA appears to have some influence on the relative atrophic response to HS, with fibers of larger CSA displaying a greater relative atrophy than fibers of smaller CSA. Similar relationships between fiber CSA and atrophy have been noted for human muscle samples obtained after spaceflight (9) and prolonged bed rest (43).
Fiber MHC isoform content and fiber Vo. MHC isoform content and fiber-type composition of the mouse soleus is strain dependent. The type I MHC isoform has been reported to comprise 70, 65, and 36% of total MHC in ICR (5), NMRI (25), and C57BL/10SnSc (25) mice, respectively. On the basis of ATPase histochemistry, type I fibers account for 75, 55, and 35% of the total soleus fibers in CBA/J (41), CD-1 (or ICR) (40), and 129B6F1/J (19) mice, respectively. In the present study, type I fibers accounted for ∼50% of total soleus MHC in male ICR and CBA/J mice and ∼20% of total soleus MHC in C57BL/6 males. Thus the composition of the ICR soleus is in good agreement with previous work, whereas the slow fiber contents of the CBA/J and C57BL/6 mice are somewhat lower than previous reports. Several factors, such as methodology (ATPase fiber typing vs. MHC identification), animal gender, and animal age, may contribute to these interstudy differences.
In the present study, 7 days of HS reduced the percentage of type I MHC from approximately one-half to one-third of total MHC in the ICR and CBA/J strains. A downregulation of type I MHC is a characteristic response of the rat soleus to HS (4, 12, 31). Our data indicate that changes in soleus MHC isoform content occur sooner in the mouse HS model compared with the rat HS model (4). However, a reduction in soleus type I MHC content is not inevitable in the mouse HS model because there was no evidence of a change in type I MHC isoform expression in the C57BL/6 mice at the whole muscle or single-fiber levels. In fact, the literature is equivocal concerning the response of mouse soleus MHC isoforms to HS. On the basis of ATPase histochemistry, 14 days of HS have been reported to decrease (26), increase (19), and have no effect (40) on the percentage of type I fibers making up the mouse soleus. With the use of electrophoretic techniques similar to those of the present study, the percentage of soleus MHC identified as type I was reported to decline from 52 to 43% after HS (3). It is noteworthy that these studies have used several different mouse strains that vary widely in terms of their soleus fiber type or MHC isoform composition under WB conditions. Taken with the present findings, it appears that there may not be a typical fiber-type response to HS for mice as there appears to be for the rat.
In addition to changes in MHC isoform expression, McDonald and Fitts (27) found that as little as 7 days of HS was associated with a >40% increase in the Vo of those rat soleus fibers that continued to express the type I MHC isoform. The average Vo of fibers obtained from the ICR strain increased by 14%. Although our statistical design did not detect a treatment effect within the CBA/J strain, the Vo of type I fibers from this strain increased by a similar amount, i.e., 16%. At this point, we consider the response of the ICR and CBA/J strains analogous to the increase in slow fiber Vo previously reported for the rat HS model, although this adaptation appears to occur either more rapidly, or to a greater extent, in the rat model.
These changes in the average Vo of fibers expressing type I MHC could be due to HS-induced alterations to proteins that influence shortening velocity, such as the myosin light chains. Ultrastructural factors, such as the geometry of the myofilament lattice (28), can have a large impact on fiber Vo. In fact, a relationship between changes in filament geometry and fiber Vo has been reported for human muscle fibers after bed rest and spaceflight (33, 34). Whether this explains the change we observed in the present mouse fibers, or whether other mechanisms are responsible or contribute, is not presently known.
In contrast to the ICR and CBA/J strains, the average Vo of the C57BL/6 type I soleus fibers was unaltered by 7 days of HS. It may be relevant that under WB conditions, the average Vo values of the type I fibers from this strain were substantially slower than expected for a species of their body mass. This appeared to be a true strain difference, because the average Vo values of type IIb fibers obtained from the C57BL/6 mice were in good agreement with predicted values, as were the Vo values of type I soleus fibers obtained from the ICR and CBA/J strains (see Fig. 4). Thus the type I fibers from the C57BL/6 mice appear to be unusual in several respects: they make up a relatively small percentage of total soleus fibers, they have a slower than predicted Vo, and they show no change in Vo with HS. McDonald and Fitts (27) identified fibers in the rat soleus that appeared unresponsive to HS; i.e., they showed no shift in Vo or ATPase activity. The authors postulated that these unresponsive fibers may have developed from primary myotubes, whereas fibers displaying a shift in Vo descended from secondary myotubes. An intriguing possibility is that the slow fibers comprising the C57BL/6 soleus differ in lineage from the slow soleus fibers of the ICR and CBA/J strains.
Unexpectedly, we found that the average Vo of fast fibers obtained from the ICR strain increased after HS. The interpretation of these data is complicated by the fact that we were not able to consistently separate the IIx and IIa MHC isoforms. The elevated average Vo of the post-HS fast fibers could, therefore, be due to a greater expression of type IIx MHC after HS, which is supported by rat models showing an increase in the IIx isoform after HS (4, 12). Improved separation of the fast MHC isoforms will be required to confirm this possibility.
Summary and implications. The major findings of this study are as follows: 1) HS resulted in significant atrophy and reductions in the Po of both slow and fast soleus fibers across all three mouse strains; 2) in all strains, specific force fell after HS, indicating that the loss in Po exceeded the degree of atrophy; 3) in all but the ICR strain, the magnitude of these changes in CSA and Po were independent of fiber MHC isoform composition, and in the ICR strain, relative changes in fiber CSA and Po may have been related to inherent differences in the CSA of the fibers under WB conditions; 4) ICR and CBA/J mice, but not the C57BL/6 strain, showed a reduction in total soleus type I MHC isoform content after 7 days of HS; and 5) HS had strainspecific effects on fiber Vo.
These data suggest that certain responses to HS, particularly those related to MHC isoform content and fiber Vo, may vary between mouse strains. Although our experiments were not designed to rigorously test these effects, they do suggest potential genetic influences on the response of skeletal muscle fibers to HS. It is well documented that behavioral and physiological responses to stress can vary widely across mice with different genetic backgrounds (24). Future studies using appropriate mouse strains could prove to be a useful method of identifying genetic factors associated with the response of skeletal muscles to HS. In contrast, the ability of fibers to produce force showed a relatively consistent change with HS across all strains studied. On the basis of a comparison of slow and fast soleus fibers, typically not possible using the rat HS model, we found that both populations of fibers experienced significant reductions in absolute and specific Po after 7 days of HS.
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