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Departments of 1Orthopedics and 2Physiology and Biophysics, College of Medicine, University of California, Irvine, California
Submitted 24 April 2006 ; accepted in final form 13 June 2007
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ABSTRACT |
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1–2%, force yields and continues to rise but with a slower slope. The resistance to stretch during the initial phase can be characterized by the stiffness of the muscle and/or the preyield modulus (Epre). Similarly, a measure of modulus also can be used to characterize the postyield modulus response (Epost). This study examined the effects of muscle atrophy and altered myosin heavy chain (MyHC) isoform composition on both Epre and Epost. Female Sprague-Dawley rats were assigned to 1) control group, 2) a hypothyroid group, 3) a hyperthyroid group, 4) a hindlimb suspension group, and 5) a hindlimb suspension + hyperthyroid group. These interventions were used either to alter the MyHC isoform composition of the muscle or to induce atrophy. Soleus muscles were stretched at strain rates that ranged from 0.15 to 1.25 muscle length/s. The findings of this study demonstrate that 4 wk of hindlimb suspension can produce a large (i.e., 40–60%) reduction in Epre. Hindlimb suspension did not produce a proportional change in Epost. Analyses of the Epre-strain rate relationship demonstrated that there was little dependence on MyHC isoform composition. In summary, the disproportionate decrease in Epre of atrophied muscle has important implications with respect to issues related to joint stability, especially under dynamic conditions and conditions where the static joint stabilizers (i.e., ligaments) have been compromised by injury. short-range stiffness; muscle atrophy; rehabilitation; ramp stretch; myosin heavy chain isoform; elastic modulus; single fiber mechanics
One approach that has been used to study the stiffness of muscles and muscle fibers during lengthening contractions is shown in Fig. 1. This approach involves fully activating a muscle or muscle fiber (i.e., allowing tension to rise onto the isometric plateau) and then imposing a ramp stretch. As shown in Fig. 1, the mechanical behavior of the muscle to a ramp stretch is quite complex. During the initial phase of stretch, force rises very rapidly; however, once the muscle or muscle fiber has undergone a strain of 1–2%, force suddenly yields such that it continues to rise but at a slower rate. The slope of the relationship between force and length (i.e., 
P/L) is known as stiffness, and some have referred to the initial slope of this relationship after the onset of lengthening as SRS (12) (see Glossary below for definitions of terms). Stiffness is sometimes referred to as a structural property because it is dependent on the dimensions of the material and its so-called intrinsic properties (11). For instance, a muscle that has a physiological cross-sectional area twice that of another muscle should have a stiffness that is approximately twofold greater. The relationship between P and L can be normalized to the dimensions of the tissue by examining the relationship between stress (force per cross-sectional area) and strain (relative length change). The slope of the stress-strain relationship is referred to as the elastic modulus or Young's modulus; because it is normalized to the dimensions of the muscle, it reflects the intrinsic or material properties of the muscle (11). In the example given above, the elastic modulus of the larger muscle should be identical to that of the smaller muscle.
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When interpreting the effects of mechanical unloading on stiffness, Epre and Epost may be confounded by transitions in MyHC isoform composition. Several findings suggest that the behavior of skeletal muscle to stretch is dependent on muscle fiber type or composition. For instance, Petit et al. (15) found that slow motor units exhibited a stiffness that was greater than that of the fast fatigable motor units. Malamud et al. (12) studied the relationship between SRS in skinned single fibers taken from the soleus and gastrocnemius muscles of cats. Beyond a strain of 1%, most of the fast-twitch type II muscle fibers exhibited an abrupt decrease in stiffness, with force continuing to rise but with a slower slope. However, slow-twitch type I fibers from the soleus and vastus intermedius muscles exhibited such a pronounced reduction in stiffness that force declined beyond the yield point and, in some instances, returned to the isometric baseline (see dashed lines in Fig. 1).
Given the above considerations, the objective of this study was to determine the extent to which SRS, Epre, and Epost are dependent on the loading state and/or MyHC isoform composition of a muscle. To achieve this objective, we manipulated the loading state and the MyHC isoform composition of the rodent soleus muscle by using the hindlimb suspension model and altering the thyroid status of the animal. Using this approach, we tested two hypotheses: 1) muscle unloading produces a loss in SRS but does not affect Epre or Epost, and 2) a slow-to-fast transition in the MyHC phenotype will reduce SRS via changes in Epre.
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250–300 g body wt) were obtained from Charles River (Wilmington, MA). Animals were randomly assigned to the following five groups: 1) control (n = 8; Con), 2) hypothyroid (n = 8; –T3), 3) hyperthyroid (n = 8; +T3), 4) hindlimb suspension (n = 8; HS), or 5) hyperthyroid plus hindlimb suspension (n = 8; HS+T3). We have used these interventions previously to manipulate the MyHC isoform composition and mechanical properties of the soleus muscle (3, 4). Animals assigned to the –T3 group underwent a thyroidectomy and were given supplemental injections of PTU every day for 4 wk. PTU was administered intraperitoneally at a dosage of 12 mg/kg. Hyperthyroidism was induced by injecting animals intraperitoneally with T3 at a dosage of 300 µg/kg every day for 4 wk. Hindlimb unloading was induced for 4 wk with the tail suspension model. The animals assigned to the HS+T3 group not only underwent 4 wk of hindlimb unloading but also received intraperitoneal injections of T3 at a dosage of 300 µg/kg every day during hindlimb unloading. We employed this combination of interventions because it has been shown to be highly effective in converting the soleus from a slow- to fast-twitch muscle (3).
Animals were housed individually and provided food and water ad libitum. In addition, each animal in the HS and HS+T3 groups was checked two to three times a day to make sure that they remained properly suspended. Approval for animal experiments was obtained from our Institutional Review Board before the experiments were conducted.
Dissection and isolation of soleus muscle for in situ contractile measurements. Animals were anesthetized with acepromazine (50 mg/kg) and ketamine hydrochloride (6 mg/kg). The hindlimb musculature was exposed by making a midsagittal incision along the entire length of the hindlimb. The skin was then freed from the underlying musculature by a blunt dissection technique. The biceps femoris was isolated and detached from its insertion along the lateral border of the tibia, exposing the sciatic nerve and the gastrocnemius muscle.
The distal tendon of the soleus and a small piece of the calcaneal insertion site were isolated. The soleus was then isolated and freed from adjacent musculature and surrounding connective tissue to allow unrestricted movement. This was performed while the blood and nerve supply were left intact. A small hook was then glued to the distal tendon of the soleus muscle insertion using cyanoacrylate glue.
The hindlimb of the animal was placed into a stereotactic frame with the femur and tibia rigidly fixed. The skin was pulled up, and a small sheet of parafilm was cut to form a muscle bath. Petroleum jelly was used to seal the parafilm against the skin. Enough mineral oil was added to completely submerge the lower hindlimb musculature. The temperature of the mineral oil was monitored and maintained at 30–30.5°C using a Digi-Sense thermocouple device (Cole-Parmer Instruments, Chicago, IL) and radiant heat from a lamp. The muscle was allowed to equilibrate for 20 min before any mechanical measurements were made.
Stimulation. In all experiments, a computer was used to control the temporal relationship of muscle stimulation, parameters of the ergometer, and data collection. The sciatic nerve was stimulated by monophasic pulses of 0.2 ms and a voltage approximately two to three times greater than the minimal voltage required to produce maximal Pt. The optimal frequency for producing Po was determined by stimulating muscles at frequencies between 75 and 150 Hz.
In situ measurement of Vo by slack test method. Measurements of Vo were made by the slack test as described previously (3). The slack test was performed to provide a mechanical index of the speed properties of the muscles in the various groups.
Slack-test measurements of the soleus muscle were conducted as follows. The muscle was maximally stimulated, allowing force to rise and reach an isometric plateau. The computer then instructed the servomotor to make length steps sufficient to cause tension to drop to zero. L was typically 2.0 mm. Each succeeding release (n = 10) was in increments of 0.2 mm. Each contraction was followed by a 1-min rest interval. Relative to Lo (mean of 32 mm), these releases corresponded to negative strains that ranged from 6 to 12% of Lo. The t associated with each quick release was defined as the time interval between the instant of release and the first indication of tension redevelopment. To achieve an unbiased determination of when tension redevelopment occurred, we used a threshold of 0.04 N. Because data collection occurred at a rate of 1,000 Hz, the resolution of determining t was 1 ms. Vo for each muscle was determined by plotting L vs. t, using a least-squares technique. Vo is the slope of the line describing the L-t relationship.
In situ measurement of force development during ramp stretches.
The force development of the soleus muscle during a lengthening contraction was measured in the following manner. Muscle length was set to Lo. The muscle was then stimulated at its optimal frequency, with force rising onto the isometric plateau. The ergometer was instructed to perform a ramp stretch 450 ms after the onset of the isometric contraction. The amplitudes of ramp stretches used in this study were 0.5, 1, and 2 mm in length. This corresponds to strains of 1.5–6% of Lo. Additionally, under each amplitude of stretch (e.g., 1 mm), the lengthening velocity was varied by completing the stretch in either 25, 50, or 100 ms. Each muscle performed a total of nine ramp stretches, using a slow-to-fast progression in the order of lengthening velocities. Collectively, the ramp velocities that were used spanned a spectrum of positive strain rates that were equivalent to 5–75% of Vo. Ramp velocities >40 mm/s were not used because in pilot studies we observed that ramp velocities greater than this resulted in a decrease in Epre when the exact conditions were repeated. The length and force signals from the ergometer were each sampled at 1,000 Hz. The muscle was allowed to rest 1 min between each contraction. Muscles were also passively stretched under each condition. The active force generated by the muscle during the stretch was simply the difference between total force minus passive force.
Determination of SRS, Epre, and Epost.
Stress was defined as the force per cross-sectional area and was expressed in units of Pascal (1 Pa = 1 N/m2). Strain was defined as equal to (L – Lo)/Lo.
To avoid subjective bias in determining SRS, Epre, Epost, y, and y, load deformation and the stress-strain records obtained from the ramp stretches were analyzed with a nonlinear regression model developed by Duggleby and Ward (6).
Muscle length measurements and estimate of physiological cross-sectional area. After the completion of the mechanical measurements, Lo (end-to-end of muscle belly) was determined with calipers. The muscle was then quickly removed from the animal, stripped of connective tissue, weighed on a Sartorius balance (resolution of 0.01 mg), and stored in 100% glycerol at –20°C. The physiological cross-sectional area was estimated with muscle mass, muscle fiber length (0.5 of Lo), and muscle density (1.05 g/cm3) (18).
Single fiber muscle mechanics. As shown in Fig. 1, Malamud et al. (12) reported that lengthening contractions of slow-twitch type I muscle fibers caused tension to initially rise very rapidly well above the isometric plateau and then yield such that tension dropped back to this initial level (i.e., the isometric plateau). We never observed such a response using the whole muscle preparation. Because whole muscle mechanics are really a composite of the individual mechanical units (e.g., single fibers), we performed a supplemental study whereby we examined the mechanical response of slow-twitch type I fibers to various ramp stretches.
Soleus muscles were removed and placed into a cold solution of relaxing buffer. Small bundles were then dissected and tied onto glass capillary tubes. These bundles were then allowed to sit in a glycerol-relaxing solution for 2–3 days. Subsequently, single fibers were isolated (2 mm in length; n = 48) and then placed in skinning solution for 5–10 min. The fiber segment was then attached to a force transducer at one end (model 308B; Aurora Scientific, Ontario, Canada) and a high-speed length controller at the other end (model 400A; Aurora Scientific) while immersed in a well containing a relaxing solution. This well was one of three milled into an aluminum plate, and the other two wells contained preactivating and activating solutions, respectively. The aluminum plate could be lowered, moved, and raised such that the solution bathing the fiber could be changed quickly. Each well had a glass bottom that allowed visualization of the fiber (with a Leica DM1L inverted microscope). In addition, a laser diffraction system was used to monitor sarcomere length (Mellos Griot, Irvine, CA). Temperature of the solution in each well was controlled at 15 or 20°C by circulating an aqueous solution through a heat exchanger in contact with the aluminum plate containing the wells. In some instances, fiber segments were tested at both temperatures to determine whether the extent of yield observed by Malamud et al. (12) was influenced by temperature. Once both ends of the fiber segment were firmly attached, the average resting sarcomere length was adjusted to a length of 2.5 µm. The fiber was then placed into the activating solution, allowing enough time for force to rise and reach an isometric plateau. The fiber segment was examined for sarcomere length uniformity and compliance at the attached ends. Those fiber segments that retained good structural organization and were rigidly fixed at their ends were then returned to the relaxing solution and tested as follows. Fiber segments were activated as described above. After rising onto the isometric plateau, the length controller was then instructed by a computer and A/D board (see above for specific details) to impose ramp stretches that varied in 1) strain (5, 7.5, and 10% Lo) and 2) duration (25, 50, or 100 ms). This combination of parameters resulted in positive strain rates that spanned from 0.5 to 4.0 fiber length/s. The fiber segment was then returned to the well with relaxing solution after completion of the ramp stretch. Two minutes later, the fiber segment underwent the identical ramp protocol but under passive conditions. This process was repeated until all combinations of strain and duration were used. During each ramp stretch, both force and length outputs were sampled at 1,000 Hz using a D/A board and computer. The active force was simply determined by subtracting the passive force record from the total force record.
The skinning solution consisted of 150 mM potassium proprionate, 5 mM KH2PO4, 5 mM magnesium acetate, 3 mM Na2ATP, 5 mM EGTA, and Triton X-100 (1% vol/vol). The relaxing solution contained the following: 100 mM KCl, 20 mM imidazole, 5 mM MgCl2, 5 mM Na2ATP, and 5 mM EGTA. The composition of the preactivating solution was identical to that of the relaxing solution, except that it contained 25 mM CrP and 300 U/ml CPK. The activating solution was identical to the preactivating solution and also contained 5 mM CaCl2. The pH of all solutions was adjusted to 7.0. The MyHC isoform composition of each fiber was determined as described previously (3).
Determination of myofibrillar protein content. Myofibril extracts of the soleus muscles were obtained using techniques described by us previously (4). All tissue preparations occurred on ice and with buffers at 4°C. Homogenized muscle was centrifuged (1,000 g, 4°C, 10 min). The biuret assay was then performed to determine myofibrillar protein concentration and content. Total myofibril yields are reported as milligrams per gram of wet muscle weight.
Determination of MyHC isoform distribution via SDS-PAGE. MyHC isoforms were electrophoretically identified with the gel system initially described by Talmadge and Roy (19) and subsequently used by us in a number of studies (3). The stacking gels were composed of 30% glycerol, 4% acrylamide-N,N'-methylene-bis-acrylamide-bis (50:1), 70 mM Tris (pH 6.7), 4 mM EDTA, and 0.4% SDS. The separating gels were composed of 30% glycerol, 8% acrylamide-bis (50:1), 0.2 M Tris (pH 8.8), 0.1 M glycine, and 0.4% SDS. The pH values of the stacking and separating gels were not adjusted after the stock solutions were mixed.
The CBS Scientific SG-200 vertical slab gel unit with the Bio-Rad 1,000/200 power supply was used for the gels. The buffers were cooled to 4°C in a refrigerator before use, and the entire gel unit was run in a cooled apparatus with a temperature below 10°C. The running time was 24 h at 275 V (constant voltage). The Bio-Rad Silver Stain Plus kit allowed visualization of the proteins. The stained gels were scanned with a Pharmacia LKB Ultrascan scanning densitometer and photographed. These were then analyzed for relative percentages of each different MyHC isoform.
Statistical analyses.
Data are reported as means ± SE. The physical characteristics of the animals and muscles reported in Table 1 were analyzed with one-way ANOVA. If a significant F-test ratio was found, then supplemental analyses were performed with a Tukey's honestly significant difference test. A similar approach was used for the contractile data reported in Table 2. Each of the variables measured during the ramp tests were initially analyzed using a two-way ANOVA. If a significant group effect was found, then the data, at each strain rate, were analyzed by one-way ANOVA and a Tukey's honestly significant difference test. The MyHC data were analyzed for each isoform by one-way ANOVA as was done for the data reported in Tables 1 and 2. The relationship between Epre and MyHC isoform composition at any given ramp velocity was examined by multiple linear regression analyses. Similarly, multiple linear regression analysis was used to determine the relationship between y and MyHC isoform composition. All statistical analyses were performed with Systat 10.2 (Systat Software, Point Richmond, CA). Statistical significance was defined as P 
0.05.
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RESULTS |
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TOPABSTRACTGlossaryMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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40% of the other three groups. The myofibrillar contents of the Con, –T3, and +T3 groups were similar, whereas those of the HS and HS+T3 groups were 20% less than those of the other groups. Muscle lengths were similar for all five groups. Speed-related properties: MyHC isoform composition and Vo. The speed-related properties of the different groups were assessed at both the protein and mechanical levels. Each of the interventions (i.e., –T3, HS, +T3, and HS+T3) produced significant alterations in the MyHC isoform profile (see Fig. 2). Relative to the Con group, the –T3 intervention resulted in a slower MyHC isoform profile, whereas HS, +T3, and HS+T3 interventions produced a faster MyHC isoform profile.
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3.4 muscle length (ML)/s. The –T3 showed an 15% decrease in Vo, whereas HS, +T3, and HS+T3 all had significant increases in Vo (see Table 2). The directional shifts in TPT are commensurate with those found in Vo. Collectively, both the protein and mechanical data demonstrate that the interventions of –T3, HS, +T3, and HS+T3 were effective in altering the speed-related properties of the soleus muscle.
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10–20 ms. After this dip, force rose in a fashion similar to that observed in Figs. 1 and 3. Because of the presence of the dip in force following yield, it was impossible to consistently determine a postyield slope for the ramp stretches that were 25 ms in duration. Hence, Epost values are not reported for such conditions.
Yield force and y.
The yield force data for the various groups are shown in Fig. 4, top. The mean yield forces of the Con and –T3 groups were very similar to each other at all strain rates. In contrast, the yield forces of the +T3 group were consistently less (about –20%) than those of either the Con or –T3 group at all strain rates. As expected, the yield forces of the HS and HS+T3 groups were dramatically less (80%) than those of the other three groups.
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y are shown in Fig. 4, bottom. There were no statistical differences in y values for the Con, –T3, and +T3 groups. The mean y values for the HS and HS+T3 groups were, however, significantly less (about –30%) than those of the Con group at all strain rates.
SRS and Epre.
The SRS and Epre of the five groups are shown in Fig. 5. SRS appeared to be a linear function of strain rate, and the slopes of the SRS-strain rate relationship appeared to be similar for the Con, –T3, and +T3 groups. The mean SRS values for the HS and HS+T3 groups were significantly less than those of the other three groups. At the slowest strain rate, the stiffness values of the HS and HS+T3 groups were 2.0 kN/m less than those of the control group. This disparity (in absolute terms) increased as a function of strain rate such that, at the fastest strain rate, the difference between these two groups and the Con group was 3.0 kN/m.
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50% (change of 6,000 kPa) less than those of the Con group at the slowest strain rate (0.15 ML/s). At the fastest strain rate, the differences between the hindlimb suspension groups (i.e., HS and HS+T3 groups) and the Con group widened such that the relative and absolute differences were 60% and 9,700 kPa, respectively.
The large differences between the Epre values of the Con and HS groups does not account for differences in specific tension. Such factors, however, can be accounted for by normalizing the preyield slope to Po. As shown in Fig. 6, there was an 35% (P < 0.001) difference in the preyield slope (Po/s) between the Con and HS groups, and this clearly demonstrates that HS produces a loss in Epre that is disproportionate to the loss in force production.
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1.25 ML/s that a significant difference from the Con value was noted for both of the HS groups. Also, note that, although Epre was clearly dependent on strain rate, no such clear strain rate dependence was evident for Epost.
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y.
The mean values for yield length and y are shown in Fig. 8. The mean values for yield length were similar among all five groups. A similar trend was found with respect to y.
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50 ms there was a substantial yield in the force record causing force to actually fall. This response occurred in all 48 fibers and under all ramp conditions. This is quite a departure from the force records obtained on whole muscle (see Fig. 1). Importantly, however, it should be noted that the single fiber experiments were performed at a much lower temperature (i.e., 15°C) than those on the whole muscle preparations (30°C). To determine whether temperature was responsible for controlling the extent of yield observed in the single fibers, additional studies were also performed at 20°C. As shown in Fig. 9, middle, temperature had a significant effect on the yield, such that at this higher temperature force continued to rise after the yield (rather than falling, as was seen at 15°C). In every fiber segment (n = 28) that was examined at the higher temperature, force always continued to rise after the yield.
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y. Second, using altered thyroid state, we were able to significantly manipulate the MyHC isoform composition of the soleus muscle and observed little if any correlation between MyHC isoform composition and Epre. Third, perhaps the single most important finding of this study was the observation that Epre of atrophied muscle was markedly less than that of normal skeletal muscle. This has important implications with respect to issues related to joint stability, especially under dynamic conditions and conditions where the static joint stabilizers (i.e., ligaments) have been compromised by injury. Surprisingly, Epost was not affected to the same magnitude as Epre, and this suggests that different mechanisms may be involved. Finally, single fiber analyses demonstrated that the postyield pattern is dependent on temperature and that the dramatic yield observed by Malamud et al. (12) in slow-twitch type I fibers is not a safety factor (as they suggested) but simply a function of the temperature at which the measurements are made. The following discussion addresses each of these findings in more detail. Is the Epre dependent on MyHC isoform composition? During lengthening contractions of activated skeletal muscle, Epre should reflect the density of attached cross bridges, the unitary stiffness of each attached cross bridge, and the magnitude of cross-bridge strain. Malamud et al. (12) commented that the Epre-strain rate relationship might be fiber type dependent because at any given strain rate slower cycling cross bridges will require a longer amount of time to detach and, as a consequence, will be strained to a greater extent. If true, then the slope of the Epre-strain rate relationship should be greater for slower muscles/muscle fibers. Malamud et al. (12), however, also commented that such a strain rate dependence might be mitigated by the possibility that, although fast cross bridges will detach at a faster rate (hence producing a lower Epre), they will also reattach at a faster rate than the slower cross bridges (hence potentially producing a greater Epre). Consistent with the former argument, Malamud et al. (12) reported that slow-twitch type I fibers had greater SRS values than fast-twitch type II fibers. In looking at the results in Fig. 5, Epre of the +T3 group was always lower than that of the Con and –T3 groups at any given strain rate. Although this finding is consistent with the concept that the Epre is fiber type dependent, a better test of this concept is provided by the slope of the Epre-strain rate relationship. In examining the slopes of the Epre-strain rate relationships for the Con, –T3, and +T3 groups, we find that it is clear that the slope of the +T3 group is similar to that of both the Con and –T3 groups (see Fig. 5). Hence, this finding strongly suggests that the Epre-strain rate relationship is not dependent on MyHC isoform composition within the context of the perturbation produced in the present study. Additionally, the slopes of the Epre-strain relationship for the HS and HS+T3 groups were very similar to each other, whereas the MyHC isoform profiles were not. This latter finding provides further support to indicate that the Epre-strain relationship is not fiber type dependent (albeit under atrophied conditions).
With respect to this issue, it should be noted that the isoform transitions in the Con, –T3, and +T3 groups were limited to the upregulation of the fast-twitch type IIA and IIX MyHC isoforms; therefore, it is unclear what influence (if any) the expression of the fast type IIB MyHC isoform would have on the Epre-strain rate relationship. Previously, our group (3) observed that 80–90% of the fibers in normal soleus muscles express only the slow type I MyHC isoform and that 4 wk of +T3 produced a significant reduction in the relative proportion of these fibers such that they only represented 10% of the total pool of fibers. Some might interpret this to indicate that the +T3 intervention produces large pools of fast fibers that are monomorphic with respect to their MyHC isoform composition. Instead, +T3 produces pools of I/IIA and I/IIA/IIX polymorphic fibers, where the percentage of the slow type I MyHC isoform represents 30–60% of the total MyHC pool. Presumably, similar changes occurred in the present study, although we did not perform such analyses. Given that these previous investigators (12) observed a relatively weak relationship between elastic modulus and muscle fiber type, one has to wonder how much of a change in elastic modulus should have been observed in the present study whereby the muscle was transformed from a slow- to a fast-twitch muscle but in a way that results in a high proportion of polymorphic fibers that express the slow type I MyHC isoform.
Mechanical unloading produces a large loss in Epre: what are the possible causes?
Stiffness is simply defined as the slope of the so-called load-deformation curve (i.e., P vs. L). As such, it is expected that muscle atrophy would result in a loss of stiffness because there are fewer sarcomeres in parallel. Some have referred to stiffness as a structural property (11), meaning that the stiffness of muscle is dependent on the architecture of the muscle and the unitary stiffness values for attached cross bridges. In contrast, elastic modulus has been referred to as a material property, reflecting the intrinsic properties of the material (11). In the case of skeletal muscle, one might hypothesize a priori that the Epre would be unaffected if mechanical unloading only reduces the number of sarcomeres in parallel. If, however, mechanical unloading also influences the function of the remaining sarcomeres, then clearly Epre should also be deleteriously affected.
In the present study, muscle mass was reduced by 40%, whereas stiffness was reduced by 80%. Hence, the greater loss in stiffness relative to that observed for muscle mass implies that something about the material properties was altered by muscle unloading. This is quantitatively described by the 40–60% loss in Epre. This observation is consistent with that of McDonald and Fitts (13) who examined the effects of mechanical unloading on the elastic modulus of single fibers taken from the rodent soleus muscle. These authors observed an 50% decrease in elastic modulus as determined by high-frequency sinusoidal length changes (5% strain at 15°C). With respect to Epre of skeletal muscle under the types of ramp stretches used in this study, there are three key factors that might account for the loss of Epre: 1) a decrease in the unitary stiffness of attached cross bridges, 2) altered excitation-contraction coupling, and 3) and altered ultrastructure.
To date, little is known about the effects of mechanical unloading on the first two factors noted above (i.e., unitary stiffness and excitation-contraction coupling). It might be suggested that the reductions in SRS and Epre simply occur because of altered excitation-contraction coupling. It should be noted, however, that the losses in SRS and Epre were disproportionate to those observed for Po and specific tension. The disproportionate reduction in Epre is emphasized further by the significant reduction in the preyield slope normalized to Po. Collectively, these observations strongly suggest that the losses in Epre must be because of changes in unitary cross-bridge stiffness and/or altered ultrastructure.
With respect to ultrastructure, there are several types of alterations that might be responsible for the loss in elastic modulus. First, there might be a number of dysfunctional myofibrils undergoing degeneration/disarray due to protein degradation. Second, thin filaments may have been altered such that their lengths are short or they are not attached to the z-line (16). Third, the lattice spacing might be increased, reducing the unitary stiffness of a given cross bridge. Fourth, there might be selective thin filament loss. Of these four factors, each has been reported to occur as a result of muscle unloading.
With respect to lattice spacing, McDonald and Fitts (13) were one of the first groups of investigators to suggest that the loss of elastic modulus was due to an increase in lattice spacing. Consistent with this concept, Riley et al. (17) reported that there was an increase in the lattice spacing of single fibers after 17 days of muscle unloading induced via bed rest.
The effects of muscle unloading on Epost are not proportional to those seen for Epre. One of the surprising findings of this study is that Epost was not affected to the same extent as Epre. This suggests that the underlying mechanisms responsible for Epre and Epost may be quite different, and, as such, Epre is much more sensitive to the atrophy that results from muscle unloading. The underlying mechanisms mediating the rise in tension following yield remain to be definitively described. Flitney and Hirst (7) suggested that cross bridges reattach with a new steady state following yield, and, as a result, force continues to rise but with a reduced slope. Alternatively, Morgan (14) proposed that yield occurs because of variability in sarcomere strength that results in variability in 1) sarcomere length, 2) cross-sectional area of the sarcomere, and/or 3) statistical variation in the numbers of attached cross bridges. Morgan suggested that yield occurs as a result of the weaker sarcomeres "popping" (i.e., a preferential lengthening of the weaker sarcomeres) and that force continues to rise after yield because the sarcomeres pop in a "weakest to strongest" order.
If the postyield response is due to cross bridges attaching at a new reduced steady state, then it seems reasonable to suggest that the Epost values of the +T3 group should have been greater than those of either the Con or –T3 groups, which it was not. This observation certainly argues against the cross-bridge hypothesis proposed by Flitney and Hirst (7). Alternatively, if the postyield response is due to sarcomere popping, then our results suggest that mechanical unloading does not significantly alter the normalized strength of the weakest-to-strongest spectrum of sarcomeres. Clearly, additional studies are needed to better clarify the underlying mechanisms responsible for the postyield force response under both normal and abnormal physiological conditions.
What is the pattern of response to ramp stretches in single fibers?
The response of whole muscle to various loading conditions is really a composite of the mechanical properties of each of individual fiber (2). During the ramp stretches employed in the present study, force initially rose very rapidly above the isometric plateau. Beyond a strain of 1–2%, the muscles typically yielded such that tension continued to rise but with a slower slope. This type of response is consistent with that observed by others (7, 9, 10, 15) but is quite different from that reported more recently by Malamud et al. (12). Malamud et al. observed that ramp stretches of fibers taken from the cat soleus muscle caused tension to rise very rapidly during the initial phase of stretch. However, beyond a strain of 1%, Malamud et al. observed that the single fibers yielded to such an extent that tension rapidly declined below the yield force.
Malamud et al. (12) hypothesized that this type of dramatic yield might represent a safety factor. Given the disparity between the single fiber observations of Malamud et al. and the whole muscle observations of the present study, we examined the response of single fibers to ramp stretches. As shown in Fig. 9, top, the response of the single fibers at 15°C was consistent with the observations made by Malamud et al. In other words, force actually declined after yield. This postyield response is quite different from that observed for whole muscle (see Fig. 1). Because the whole muscle measurements were made at 30°C, we examined the influence of temperature on the single-fiber response. As shown in Fig. 9, middle, the postyield response is quite dependent on temperature, and the responses observed at 20°C were consistent with those seen for whole muscle. Hence, we would suggest that the postyield responses observed by Malamud et al. (see also dashed line in Fig. 1) do not represent a safety feature but instead represent the influence of temperature on cross-bridge behavior following yield. It seems reasonable to suggest that, at low temperatures, slow rates of cross-bridge attachment and force development will lead to a significant reduction in force after yield, producing a response similar to that shown in Fig. 9, top. In contrast, at higher temperatures where the rates of cross-bridge attachment and force development are faster, cross bridges should reattach quicker, minimizing the extent of yield (see contrast between top and middle of Fig. 9).
Potential clinical significance. Ligaments are often referred to as static joint stabilizers, whereas skeletal muscles are thought of as dynamic stabilizers. Ligamentous injuries are quite common, especially among those participating in organized and recreational athletics. Often, these types of injuries require surgical repair, and skeletal muscle atrophy is a common consequence of such intervention. Clearly, the stiffness of skeletal muscle during lengthening contractions is due, in part, to its physiological cross-sectional area. Hence, muscle atrophy that occurs after ligamentous injury and surgical repair decreases the stiffness of that muscle and its ability to protect the joint ligaments during activity. With respect to the findings of this study, however, the loss of stiffness is not simply proportional to the loss in cross-sectional area. Rather, our findings clearly demonstrate that mechanical unloading can also affect stiffness by altering the material properties of skeletal muscle, resulting in large losses of Epre (i.e., a reduction in stiffness normalized to the geometry of the muscle; elastic modulus).
Presently, the dependence of injury on muscle stiffness remains to be determined. However, it is interesting to note that female athletes are more susceptible to ligamentous injuries of the knee than male athletes, and this appears to be associated with weaker knee extensor muscles (1, 20).
Important technical considerations and limitations.
There are several technical issues that should be noted. First, the advantage of using a muscle like the soleus is that it has a very high degree of plasticity, which is uncommon among rat hindlimb muscles. This is a significant consideration given that different MyHC isoform profiles can be studied under identical architectural conditions. Hence, the rat soleus muscle represents a good experimental model for exploring issues related to MyHC isoform composition and whole muscle mechanics. Second, the strain rates used in the present study were relatively slow, with a maximum of 1.25 ML/s. Hence, further studies that employ a faster spectrum of strain rates are needed to conclusively demonstrate whether the Epre-strain rate relationship is dependent on MyHC isoform composition. We used relatively slow strain rates, because in a pilot study (unpublished observations) our group observed that faster strain rates actually produced a form of injury whereby there was a minor reduction in Po (5–10%) that was accompanied by much larger losses in Epre (20–25%). Finally, it should be noted that all measurements on whole muscle were performed at 30°C. The reason for this is that we wanted to use conditions that maintained the viability of the muscle preparation and yet represented a temperature that was somewhat close to the resting peripheral temperature of 35°C.
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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.
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