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J Appl Physiol 84: 229-235, 1998;
8750-7587/98 $5.00
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Contractile function of single muscle fibers after hindlimb unweighting in aged rats

L. V. Thompson and J. A. Shoeman

Program in Physical Therapy, University of Minnesota, Minneapolis, Minnesota 55455

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Thompson, L. V., and J. A. Shoeman. Contractile function of single muscle fibers after hindlimb unweighting in aged rats. J. Appl. Physiol. 84(1): 229-235, 1998.---This investigation determined how muscle atrophy produced by hindlimb unweighting (HU) alters the contractile function of single muscle fibers from older animals (30 mo). After 1 wk of HU, small bundles of fibers were isolated from the soleus muscles and the deep region of the lateral head of the gastrocnemius muscles. Single glycerinated fibers were suspended between a motor lever and force transducer, functional properties were studied, and the myosin heavy chain (MHC) composition was determined electrophoretically. After HU, the diameter of type I MHC fibers of the soleus declined (88 ± 2 vs. 80 ± 4 µm) and reductions were observed in peak active force (47 ± 3 vs. 28 ± 3 mg) and peak specific tension (Po; 80 ± 5 vs. 56 ± 5 kN/m2). The maximal unloaded shortening velocity increased. The type I MHC fibers from the gastrocnemius showed reductions in diameter (14%), peak active force (41%), and Po (24%), whereas the type IIa MHC fibers showed reductions in peak active force and Po. Thus 1 wk of inactivity has a significant effect on the force-generating capacity of single skeletal muscle fibers from older animals in a fiber type-specific manner (type I MHC > type IIa MHC > type I-IIa MHC). The decline in the functional properties of single skeletal muscle fibers in the older animals appears to be more pronounced than what has been reported in younger animal populations.

contractile properties; inactivity; fiber types; myosin

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE BIOCHEMICAL and physiological properties of limb skeletal muscle adapt to a variety of experimental conditions (10, 12, 14, 15, 21, 26). One condition is the absence of normal weight-bearing activity. Inactivity causes muscle atrophy, with the greatest change observed in the antigravity muscles such as the soleus of adult animal populations (7, 10, 12, 31). One frequently used model for the study of the cellular and molecular mechanisms responsible for inactivity-induced changes is hindlimb unweighting (HU). HU produces whole skeletal muscle atrophy and single skeletal muscle fiber atrophy, enzymatic and contractile property changes, and reduced peak power (7, 10, 14, 17, 21, 24, 26). Single slow-twitch type I fibers show the greatest change after HU (12).

Research studies examining the older animal have investigated skeletal muscle contractile properties at the whole muscle level to determine isometric and shortening contractions, twitch properties, and maximal unloaded shortening velocity (Vo; 3, 5, 18). Recently, the contractile properties of single skeletal fibers and myosin heavy chain (MHC) isoform compositions from the soleus (Sol), extensor digitorum longus (EDL), and gastrocnemius (Gast) muscles from older rats were characterized (2, 19, unpublished observations).

Although the effects of HU on skeletal muscle from adult animals have been examined, the skeletal muscles in the limbs of older rats have received relatively little attention (30-mo-old animals) (20). The degree of functional change after HU in individual slow- and fast-twitch fibers is unknown in an animal population that is older. Understanding the adaptation of skeletal muscle to short-term inactivity in an older animal population is important for developing appropriate countermeasures.

The specific purpose of the present study was to examine the effects of 1 wk of inactivity (short term) on the functional properties of single skeletal Sol and Gast muscle fibers. The single skeletal fibers expressed the type I MHC isoform, the type IIa MHC isoform, or both the type I and type IIa MHC isoforms. We hypothesized that single fibers from the skeletal muscles of older rats would demonstrate fiber type-specific alterations after HU (type I MHC > type IIa MHC). A single glycerinated-fiber preparation was chosen to study fiber type contractile properties [force-generating capacity, Vo, and MHC composition]. This preparation allows investigation of the function of myofilament proteins in a cell with an intact filament lattice but without the confounding effects related to intercellular connective tissue or protein heterogeneity between cells of multicellular preparations.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animal care and HU. An animal care protocol was approved by the Institutional Animal Care and Use Committee and the guidelines of the National Institutes of Health. Nineteen 30-mo-old Fisher 344 Brown Norway F1 hybrid rats (FBN) were purchased from the National Institute on Aging and placed in individual cages (20). Thirty months of age was chosen because it is the age of ~50% mortality of a litter (20). The cages were housed in a separate room in a conventional animal facility under a 12:12-h light-dark cycle at 20°C, and the animals were maintained on a diet of Rodent Chow and water ad libitum. After 1 day of acclimation to the environment, the randomly assigned HU animals were partially unweighted for 1 wk with a harness attached to the proximal two-thirds of the tail (2). The animals were in the animal facility for 8 days and showed no signs of inflammation or disease.

Solutions for single glycerinated fibers. The composition of all the solutions used in the single glycerinated (sarcolemma chemically disrupted)-fiber experimental protocols was determined by using a computer program (9). The relaxing and activating solutions contained the following (in mM): 7.0 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 5.4 MgCl2, 20 imidazole (pH 7.0), 14.5 creatine phosphate, 4.7 ATP, as well as 150 U/ml creatine phosphokinase, and CaCl2 to achieve pCa 9.0 (-log[Ca2+]; relaxing solution) or pCa 4.5 (activating solution). All solutions contained enough KCl to achieve an ionic strength of 180 mM and were adjusted to pH 7.0 with KOH. The glycerinating solution contained (in mM) 125 K-propionate, 2 EGTA, 4 ATP, 1 MgCl2, and 20 imidazole (pH 7.0) as well as 50% glycerol (vol/vol) and the protease inhibitor leupeptin (0.1%, final concentration). Ringer solution contained (in mM) 95 NaCl, 2.5 KCl, 1 MgCl2, 1.8 CaCl2, and 25 NaHCO3.

Muscle and single-fiber preparation. After 1 wk of HU, the experimental and control animals were weighed and anesthetized with pentobarbital sodium (35 mg/kg body wt, ip). The Sol, lateral and medial heads of the Gast, plantaris, EDL, and tibialis anterior muscles were isolated, rinsed with cold Ringer solution (4°C), trimmed free of excess fat and connective tissue, and weighed (2). The Sol muscles of the left hindlimbs were then placed in cold (4°C) relaxing solution. Nine bundles (~50 fibers) were isolated from the Sol muscle (source of slow-twitch type I fibers). The bundles were stretched to approximately in situ length and tied to glass capillary tubes with 4-0 surgical suture, placed in glycerinating solution, and stored at -20°C for up to 4 wk. The Gast muscles of the left hindlimbs were then placed in cold relaxing solution, the deep portion of the lateral head [portion of the Gast muscle defined as the red gastrocnemius (RG)] was dissected free, and 12 bundles were formed and placed in glycerinating solution.

Single-fiber experimental protocol. On the day of the experiment, a glycerinated fiber bundle was placed in a dissection chamber containing relaxing solution (4°C), and 5-10 single glycerinated fibers were isolated. One isolated fiber was randomly selected and transferred to an experimental chamber containing relaxing solution. Two to nine fibers per muscle were evaluated. The temperature-controlled chamber was mounted to the stage of an inverted microscope equipped with a Panasonic camera. The chamber included a spring-mounted stainless steel plate insert with three wells containing activating or relaxing solution. The bottom of each well was sealed with a glass coverslip that allowed the mounted fiber to be transluminated for viewing. The fiber (2-3 mm long) was attached by tweezers between an isometric force transducer (Cambridge model 403, Cambridge, MA; sensitivity 2 mV/mg) and an arm of a servo-controlled direct current torque motor (Cambridge model 300H, Cambridge, MA) (2). The fiber was bathed in relaxing solution containing 0.5% (wt/vol) Brij-58 (polyoxyethylene 20 cetyl ether; Sigma Chemical, St. Louis, MO) to inhibit any remaining sarcoplasmic reticulum Ca2+ uptake activity. Any fiber showing a high degree of striation nonuniformity or a damaged region was discarded.

Determination of fiber diameter and optimal length. The single glycerinated fiber was observed through a Nikon inverted microscope (×600) and with a Panasonic video camera system. The segment length was adjusted to a sarcomere spacing of 2.5 µm in relaxing solution by using a calibrated eyepiece. The fiber diameter was determined as the average of three measurements made (by using the calibrated eyepiece) along the length of the fiber while the fiber was in relaxing solution. The fiber cross-sectional area (CSA) was calculated by assuming a circular cross section. Previous work has reported a 20 or 50% swelling of fiber diameter after the removal of the sarcolemma; therefore, a permeabilized fiber with a diameter of 88 µm corresponds to 58 µm (8, 13). Data were reported without any correction for swelling.

Segment length was determined by moving the microscope stage with a micrometer such that the fiber segment moved across the visual field of the eyepiece. The segment length was determined directly from the micrometer displacement. All experiments were conducted at 15°C.

Determination of peak active force and peak specific tension (Po ). The outputs of the force and position transducers were amplified and sent to a PC microcomputer via a universal input-output board (LabMaster, 100-kHz analog-to-digital converter) and to an oscilloscope (Nicolet 310) for immediate examination. Data were collected, analyzed on-line, and stored by customized software. Baseline was monitored in relaxing solution (pCa 9.0), and the fiber was activated by transfer into activating solution (pCa 4.5) (Fig. 1). Peak active force (in mg) was determined in each fiber by computer subtraction of the baseline from the peak active force (2). Po (kN/m2) was calculated from the peak active force and fiber CSA. A Po of 77 kN/m2 corresponds to 116 kN/m2 when corrected for fiber swelling. Po values were reported without correction for fiber swelling. In the present study all data were eliminated when the peak active force dropped below 80% of the initial value.


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  		Fig. 1.   Original single glycerinated-fiber force record. pCa 4.5 activating solution produced peak active force. Spike at beginning of activation results from movement of fiber between relaxing and activating solutions. Bar, 10 mg.

Determination of Vo . Vo of individual fibers was determined by the slack test method (2). The fiber was transferred to the activating solution, and force was allowed to develop. Slack (Delta L) was rapidly introduced into the fiber by movement (complete within 2 ms) of the torque motor arm when the developed force reached a plateau. The time interval (Delta t) between the instant at which Delta L was introduced and when force started to redevelop was measured directly by using the computerized program. Vo was determined as the slope of the linear regression of Delta L against Delta t (Fig. 2).


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  		Fig. 2.   Determination of single-fiber maximal unloaded shortening velocity (Vo). Slack step length (Delta L) was plotted against time required for redevelopment of force (Delta t), with slope of best fit line defining Vo and normalized for fiber length (FL; in FL/s). Myosin heavy chain (MHC) composition of fibers was subsequently determined by SDS-PAGE. Representative single fibers are the following: Control type IIa MHC with a Vo of 3.6 FL/s (black-triangle), hindlimb unweighted group (HU) type I MHC from soleus (Sol) muscle with a Vo of 1.71 FL/s (black-square), and a Control type I MHC from Sol muscle with a Vo of 0.74 FL/s (bullet ).

Fiber type identification and myosin analysis. After the physiological measurements were made, the MHC composition of each fiber (2 mm) was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (2). The fibers were solubilized in 10 µl of 1% SDS sample buffer and stored at -80°C. Approximately one-half of the volume (1-mm fiber segments) was loaded on gels, consisting of a 3% (wt/vol) acrylamide stacking gel and a 5% separating gel for MHC analysis (Fig. 3).


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  		Fig. 3.   Representative 5% polyacrylamide gel demonstrating MHC identification in single skeletal muscle fibers. Lanes 1, 2, and 3: standards representing type IIb MHC, both type IIa and type I MHC, and type I MHC fibers, respectively; lanes 4-6, Control red gastrocnemius (RG); lanes 7-9, HU RG; lanes 10-11 Control Sol; and lane 12, HU Sol.

Statistical analysis. For statistical comparisons, the body weights, muscle wet weights, and muscle wet weight-to-body weight ratios in the different experimental conditions (Control and HU) were evaluated by one-way analysis of variance (ANOVA) to determine whether any statistically significant differences occurred. If such differences were present, the Tukey honestly significant post hoc multiple-comparison test was used to compare the Control and HU groups by body weight or muscle type. The level of significance was set at P = 0.05.

In this study 175 individual skeletal fibers were examined. The number of individual fibers per fiber type and experimental condition ranged from 17 to 29. The individual fiber data values (diameter, peak absolute force, Po, and Vo) were meaned by fiber type (type I, type IIa, and type I-IIa MHC) and muscle source (Sol, RG) per animal. A mean for all fibers of a specific MHC type within 1 muscle for 1 observation resulted in 10 Control observations and 9 HU observations. Subsequently, comparisons between the diameter, peak active force, Po, and Vo of Control and HU groups by fiber type and muscle source were evaluated by the ANOVA. The level of significance was set at P = 0.05. If the F-statistic of the ANOVA was significant, the Tukey honestly significant difference multiple-comparison tests for unequal group sizes was used to test the difference between Control and HU groups by fiber type and muscle source.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of HU on muscle mass. Compared with the Control rats, HU rats displayed reductions between 11 and 23% in absolute muscle masses, with the Sol and Gast muscles demonstrating the greatest reductions and the EDL the least reduction (Table 1). The relative muscle masses (muscle mass/body mass) decreased between 11 and 19%. The muscles that are involved with plantarflexion of the ankle joint (Sol, Gast, plantaris) showed a greater decline in both absolute muscle mass and relative muscle mass than the muscles involved with dorsiflexion of the ankle joint (EDL, TA).

                              
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  		Table 1.   Animal characterization for 30-mo-old Fisher 344 Brown Norway F1 hybrid rats

Effect of HU on diameter and force-generating capacity of single fibers. Single-fiber diameters, peak active force, and Po (mg/CSA) are summarized in Table 2 by fiber type and muscle source. Consistent with the whole muscle atrophy, a reduction of 14% in the diameter of the type I MHC fibers from the Sol and RG was observed. Single-fiber force production was significantly reduced as a result of fiber atrophy, as indicated by 41% (RG type I MHC fibers) and 40% (Sol type I MHC fibers) drop in peak active force. Po also fell 24 and 30% after HU in RG type I MHC and Sol type I MHC fibers, respectively, indicating that the atrophied fibers had a reduced ability to produce force per unit CSA. Figure 4 shows the HU-induced shift in Po. For example, after HU, 56% of the type I MHC fibers displayed a Po <=  60 kN/m2, whereas only 24% of the type I MHC fibers in the Control group displayed a Po <=  60 kN/m2. Peak active force and Po significantly declined by 38 and 29%, respectively, after HU in fibers expressing type IIa MHC, whereas fiber diameter of this fiber type population did not change from Control.

                              
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  		Table 2.   Diameter, peak active force, and peak specific tension of single skeletal fibers


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  		Fig. 4.   Frequency distributions of peak specific tension (Po) for Control Sol type I MHC (A) and HU Sol type I MHC fibers (B). Two to nine fibers were studied per muscle (1 fiber/bundle).

Effect of HU on Vo . Table 3 shows mean values ± SE for the fiber Vo. The Vo of Control fibers expressing type I MHC from Sol muscle was 0.94 ± 0.08 fiber lengths (FL)/s and ranged from 0.27 to 1.82 FL/s. The mean Vo of type I MHC from the HU Sol muscle was 42% faster than the Control mean. Figure 5 represents the Vo distribution. After HU, 23% of the type I MHC fibers displayed a Vo >=  1.81 FL/s, whereas only 4% of the Control group of type I MHC fibers had a Vo >=  1.81 FL/s. The type I MHC fibers from the RG showed a tendency to increase; however, it was not significant at the P < 0.05 level. One week of HU induced a 33% increase in the Vo of the fibers coexpressing type I and type IIa MHC (2.28-3.39 FL/s); in contrast, the Vo in type IIa MHC fibers showed no change.

                              
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  		Table 3.   Maximal shortening velocity of single skeletal fibers


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  		Fig. 5.   Frequency distributions of Vo for individual fibers from Control Sol type I (A) and HU Sol type I MHC fibers (B). Fibers were determined by MHC profile. Two to nine fibers were studied per muscle (1 fiber/bundle).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The major findings of the present study were significant decreases in Sol-to-body mass ratio, fiber diameter, peak active force, and Po and an increase in Vo in type I MHC fibers from the Sol muscle of an older animal population. The type I MHC fibers from the RG responded to HU in a pattern similar to that in the type I MHC fibers of the Sol muscle. In contrast to previous HU studies in adult animal populations and in preliminary data of 12-mo-old FBN rats, the type IIa MHC fibers were not resistant to HU and showed significant decreases in the peak active force and Po. The single fibers coexpressing both type I and type IIa MHC were resistant to HU. The findings suggest significant alterations in the force-generating capacity of single fibers to short-term inactivity in the older animal population, which are fiber type specific.

Muscle mass, single-fiber diameter, and HU. Previous literature investigating HU-induced changes in muscle mass has demonstrated a preferential atrophy between the predominantly "slow" Sol muscle and the predominantly "fast" Gast muscle, with the Sol more sensitive to HU (1, 7, 10, 24). In older animals (30 mo old), the Sol muscle continues to be composed of predominantly type I MHC fibers, and the HU-induced response is not surprising (16, unpublished observations). However, in the older animal the HU-induced atrophy is also observed in the Gast muscle. The Gast muscle in the older animal is composed of a mixed population of fibers types (type I, type IIa, and type IIb MHC and fibers coexpressing both type I and type IIa MHC) (27, unpublished observations). The percentage of fibers coexpressing both type I and type IIa MHC increases with age, and the altered fiber type composition of the Gast muscle appears to have an influence on the extent of sensitivity to HU (27).

A preferential atrophy is also observed at the single-fiber level in the older animal. The observed HU-induced differential effect on single-fiber diameter (type I MHC fiber > type IIa MHC fiber atrophy or type I-IIa MHC fiber) is also reported in the 12-mo-old FBN rat and other rat strains (12, 15, 16). The literature investigating HU-induced changes has generally examined longer periods of HU (7, 10, 12, 14). In the present study, HU was for only 1 wk, making comparison between the studies difficult; however, the results suggest that the HU-induced changes at the whole muscle level and at the single-fiber level are similar or greater than what has been reported in younger animal populations (16, 21).

The mechanisms underlying the selective atrophy may be related to protein synthesis and degradation rates (11, 14, 28, 31). Garlick et al. (11) demonstrated a decline in protein synthesis in all muscles with increasing age, mostly as a result of a fall in the RNA content. They also observed that the rates of protein turnover in the Sol muscle were higher than those in other muscles and suggest a relationship between high rates of protein synthesis and the content of slow oxidative fibers (11). In rats, at the whole muscle level, an age-related fast-to-slow myosin isoform transformation has been observed, resulting in an increase in slow myosin isoforms (unpublished observations). Thus the differential atrophy at the whole muscle level and single-fiber level seen with HU in the older animal may be related to the increased rates of protein turnover in the fibers with slow myosin isoforms.

Studies have provided evidence that increased protein breakdown may also play a role in muscle atrophy (30, 31). Taillandier et al. (30) demonstrated that both the lysosomal and the Ca2+-activated proteinases contribute to the increased muscle proteolysis seen with HU. They also suggest that increased ATP-ubiquitin-dependent proteolysis is mainly responsible for the wasting of the Sol muscle with HU (30). Further research is needed to determine which pathway may play a role in the muscle wasting of the older animal and whether protein breakdown is muscle specific.

Force and HU. In adult animal populations, HU is known to reduce the ability of the whole Sol muscle to produce force (6, 10). Single-fiber studies have found peak active force to be reduced after HU; however, the reduction in single-fiber force is not due solely to gross fiber atrophy, because peak active force declines to a greater extent than does diameter (12, 21, 24). This finding is expressed as a decline in Po, or the force produced per CSA of fiber (12, 21, 24). In adult FBN rats, HU results in a 24% decline in peak active force and a 16% decline in Po of type I MHC fibers from the Sol muscle (16). McDonald and Fitts (21) found a similar change in Po after 1 wk of HU by using a different strain of rat. The findings of the present study indicate that the single skeletal muscle fibers from the older animal are more sensitive to 1 wk of inactivity. The reported 40% decline in peak active force and 30% decline in Po in the type I MHC fibers after 1 wk of HU are greater than what has been reported in the younger animal population. The findings suggest that the force-generating capacity in single cells is very susceptible to removal of weight bearing in the older animal population.

The present study demonstrated that the Po of type I MHC fibers and of the type IIa MHC fibers from the RG in the older animal are also altered with HU, whereas the Po of fibers coexpressing type I and type IIa MHC did not change. The extent of decline in Po of the type I MHC fibers from the RG muscle is similar to the extent of decline in Po of the type I MHC fibers from the Sol muscle. This finding suggests that the removal of weight bearing affects the type I MHC fibers in muscles of the hindlimb with different fiber type compositions. The selective resistance of the fiber coexpressing both MHC isoforms (type I and type IIa) with HU may be a result of function.

The mechanism for the decline in Po may be attributable to a decrease in myofibrillar protein concentration, and direct evidence for this possibility has been shown by Thomason et al. (31). Again, altered protein synthesis and breakdown rates may be the underlying mechanism for a decrease in protein concentration.

The Po values reported from the skinned fibers tended to be lower than those previously reported in the literature (21). This difference would be less if correction were made for the fiber swelling that occurs during skinning. Correction of CSA for this swelling would bring Po values for the skinned fibers into the range of 120 kN/m2. We have reported skinned fiber Po values in 12-mo-old FBN animals at 92 kN/m2 and, after correction of CSA for swelling, the mean Po value would be 139 kN/m2. The reason for the tendency toward lower values is unknown; however, we have maintained consistency between experimental groups when collecting data (solutions, temperature, diameter measurements). It is noteworthy to highlight that all the single-fiber diameter measurements were determined while the fibers were in solution, which may result in overestimation of the diameter. The overestimation would then provide a reason for the lower values.

Fiber Vo and HU. Vo of limb skeletal muscle is hypothesized to be dependent on the rate of ATP hydrolysis by the actomyosin adenosinetriphosphatase (ATPase) (4). This is supported by findings of a significant correlation between Vo and myofibrillar ATPase in limb skeletal muscle (6, 21, 26). HU caused a shift in fiber Vo intermediate between slow-twitch type I and fast-twitch type IIa fibers. The cellular basis for this functional change is unknown. All Sol fibers in this study expressed type I MHC on SDS-polyacrylamide gel electrophoresis. A portion of fibers in this study had HU-induced elevated Vo and the same myosin protein profile as the Control group. The range of Vo for all (Control and HU) type fibers is 0.27-2.86 FL/s. Reiser et al. (23, 25) have shown a strong quantitative correlation between Vo and MHC composition in single rabbit and chicken fibers. The observed 42% increase in Vo of single fibers expressing type I MHC in older rats after 7 days of HU is significant because previous observations in adult animals have only reported a 32% increase (21). Although the mechanism responsible has not been identified, the elevated Vo does not appear to be due to undetected levels of fast MHC in single fibers classified as type I. Several explanations could be HU-induced change in myosin light chain stoichiometry, myosin transformation to an intermediate myosin (type IIx MHC) not detected in the present study, or alterations in regulatory proteins (22, 29).

In summary, the force-generating capacity of single skeletal fibers (type I and type IIa MHC) is altered significantly after HU in the older animal population. The single skeletal fibers coexpressing both type I and type IIa MHC are more resistant to HU-induced atrophy in the older animal population. The HU-induced alterations are fiber type specific and appear to be greater than what has been reported in younger animals.

    ACKNOWLEDGEMENTS

The authors thank Katie Alley and Mike Sandmann for technical support.

    FOOTNOTES

This study was funded in part by the American Heart Association, Minnesota Affiliate, the Minnesota Medical Foundation, and a University of Minnesota Graduate School Grant-in-Aid.

Address for reprint requests: L. V. Thompson, Dept. of Physical Medicine and Rehabilitation, Univ. of Minnesota, Minneapolis, MN 55455 (E-mail: Thomp067{at}maroon.tc.umn.edu).

Received 5 December 1996; accepted in final form 20 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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The Journal of Applied Physiology 84(1):229-235
0161-7567/98 $5.00 Copyright © 1998 the American Physiological Society


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