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Am J Physiol Cell Physiol 295: C249-C256, 2008. First published May 28, 2008; doi:10.1152/ajpcell.90640.2007
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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Force deficits and breakage rates after single lengthening contractions of single fast fibers from unconditioned and conditioned muscles of young and old rats

Gordon S. Lynch,1 John A. Faulkner,2 and Susan V. Brooks2

1Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, Victoria, Australia; and 2Departments of Molecular and Integrative Physiology and Biomedical Engineering, The University of Michigan, Ann Arbor, Michigan

Submitted 19 December 2007 ; accepted in final form 22 May 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The deficit in force generation is a measure of the magnitude of damage to sarcomeres caused by lengthening contractions of either single fibers or whole muscles. In addition, permeabilized single fibers may suffer breakages. Our goal was to understand the interaction between breakages and force deficits in "young" and "old" permeabilized single fibers from control muscles of young and old rats and "conditioned" fibers from muscles that completed a 6-wk program of in vivo lengthening contractions. Following single lengthening contractions of old-control fibers compared with young-control fibers, the twofold greater force deficits at a 10% strain support the concept of an age-related increase in the susceptibility of fibers to mechanical damage. In addition, the much higher breakage rates for old fibers at all strains tested indicate an increase with aging in the number of fibers at risk of being severely injured during any given stretch. Following the 6-wk program of lengthening contractions, young-conditioned fibers and old-conditioned fibers were not different with respect to force deficit or the frequency of breakages. A potential mechanism for the increased resistance to stretch-induced damage of old-conditioned fibers is that, through intracellular damage and subsequent degeneration and regeneration, weaker sarcomeres were replaced by stronger sarcomeres. These data indicate that, despite the association of high fiber breakage rates and large force deficits with aging, the detrimental characteristics of old fibers were improved by a conditioning program that altered both sarcomeric characteristics as well as the overall structural integrity of the fibers.

contraction-induced injury; permeabilized single fibers; aging

MAXIMALLY ACTIVATED MUSCLE fibers are not injured by fixed-end (isometric) contractions, or when shortening, but are injured during contractions when fibers are stretched (33, 34). Such "lengthening contractions" occur whenever the load on the contracting muscle exceeds the force developed by the muscle, as happens during running down stairs or down hills or during falls (12). Contraction-induced injury is of greater significance for older than for younger persons, because skeletal muscle fibers in old animals are more easily injured and, for a given lengthening contraction protocol (LCP), sustain a greater injury (6, 8, 9, 52). In addition, when of sufficient severity, the injury may result in permanent loss of muscle mass and force in old animals (5, 41, 42). Furthermore, the incidence of sudden falls increases dramatically in the elderly due to age-related losses in muscle mass and strength (49), with consequences for movement and stability (16).

Both contraction-induced injury and fatigue contribute to the force deficit observed immediately after muscles of humans (13, 38), rats (1, 47, 48), or mice (9, 33) have been stretched repeatedly during maximum, or near maximum, activation. The recovery from fatigue occurs within the first hour (33), but depending on the severity of the contraction-induced injury, the force deficit may persist for weeks (33), or even longer (5, 41, 42). For investigations of lengthening contraction-induced injury, the confounding factor of the contribution of fatigue to the magnitude of the force deficit during repeated contractions is eliminated by protocols of single stretches of maximally activated whole muscles (9, 10). Such protocols may also be replicated with single stretches of maximally activated permeabilized single fibers from mice (6, 7), rats (25, 30, 31), or humans (46). Following single lengthening contractions of up to 20% strain [stretched by 20% of fiber length (Lf)], force deficits were as much as twofold greater for permeabilized single fiber segments from extensor digitorum longus (EDL) muscles of old compared with young control rats, termed "young-" and "old-control" fibers (6). Despite the greater force deficits reported for old- compared with young-control fibers, high rates of fiber breakage have been reported for permeabilized single fibers from vastus lateralis muscles of elderly humans (45). Such breakages may constitute a confounding variable in the comparison of force deficits of fibers from animals of different ages. Fibers prone to breakage likely have high values for force deficit at a given strain, but breakage removes fibers from the sample on which force deficits are measured. The numbers of breakages are almost never reported in studies of permeabilized single fibers.

Our primary purpose was to compare force deficits and frequency of fiber breakages for young and old muscle fibers to better understand the mechanisms underlying age-related deficits in muscle fiber function and the protection conferred on muscle fibers by repeated bouts of lengthening contractions. Conditioning with LCPs produces major adaptations in susceptibility to injury of whole muscles of young and old mice (8) and in the isometric contractile properties of permeabilized single fibers of older humans (14, 46). Conditioning with LCPs has the potential to reduce both force deficits and breakages of permeabilized single fibers, but this potential has not been investigated previously. Three hypotheses were tested using single stretches of maximally activated permeabilized single fibers: 1) for stretches of young-control fibers of up to 30% strain, breakages are few and have no effect on force deficits; 2) with increasing strain, young-control and old-control fibers develop greater force deficits, but the magnitude of the difference in force deficit between young and old fibers decreases as increasing numbers of old fibers develop breakages; and 3) force deficits and number of breakages do not differ between "young-conditioned" and "old-conditioned" fibers.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experiments were performed on 158 permeabilized single fibers obtained from control EDL muscles of six young (6 mo old) and six old (26 mo old) male Fischer 344 rats, which provided the fibers termed young-control and old-control fibers, respectively. An additional 75 fibers designated as young-unconditioned and old-unconditioned and young-conditioned and old-conditioned fibers were obtained from separate groups of three young and three old conditioned male Fischer 344 rats. Conditioned fibers were obtained from EDL muscles that had experienced 6 wk of lengthening contractions, and unconditioned fibers were obtained from the contralateral EDL muscles. Throughout the experimental period, each of the rats remained cage-sedentary in specific pathogen-free facilities and was fed standard laboratory chow ad libitum. All experiments were conducted in accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996) and were approved by the University Committee for the Use and Care of Animals at The University of Michigan. The excision of the muscles for preparing single fibers was performed with the rat under deep pentobarbital sodium anesthesia. Following the removal of the tissue, with the rat still under deep anesthesia, the rat was euthanized by an overdose of the anesthetic, and the thoracic cavity was opened to ensure a humane death.

Conditioning of skeletal muscles with the LCP. The right dorsiflexor muscles were exposed to an in vivo LCP once a week for 6 wk. The LCP was administered on a modification of the "shoe-apparatus" designed to measure the mechanical behaviors of dorsi- and plantarflexor muscles of the ankles of mice in vivo (2) and described for the conditioning of the dorsiflexor and EDL muscles of mice (8). Briefly, the rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (100 mg/kg for 8- to 10-mo-old rats and 75 mg/kg for 20- to 22-mo-old rats), with supplemental doses administered as required to maintain an adequate level of anesthesia. Rats were placed on a Plexiglas platform maintained at 37°C, the right knee was secured between sharpened screws, and the right foot was secured in the shoe apparatus. The shoe fixture was attached to a torque transducer (model QWFK-8M, Sensotee, Columbus, OH) mounted on the shaft of a custom-built servomotor. The apparatus was designed to produce accurate rotations of the ankle joint. Control of the motor and collection of data from the torque transducer were performed using LabView software (National Instruments, Austin, TX).

The dorsiflexor muscles were activated through stimulation of the peroneal nerve with a pair of needle electrodes. Stimulation voltage, frequency, and ankle angle were adjusted for maximum isometric torque. Maximally activated muscles were exposed to a protocol of 180 lengthening contractions performed at 0.20 Hz for three 5-min bouts, with a 5-min rest between each bout of contractions. The first 100 ms of each contraction was isometric at optimal ankle angle. For the final 700 ms of each contraction, the ankle was rotated through 40° at a rate of ~60° per second. When not performing the weekly LCP, the rats remained sedentary in their cages with free access to standard laboratory chow and water. They did not perform any other exercise apart from the LCP and did not have access to activity wheels or similar exercise devices.

Within a week of the completion of the 6-wk conditioning program, the right EDL muscle was evaluated in situ to demonstrate that the muscle was, in fact, conditioned. EDL muscles in anesthetized rats were exposed to an in situ protocol of 225 lengthening contractions of 20% strain. After 3 days, rats were once again anesthetized, and maximum isometric force of the right EDL muscle was measured in situ. In dramatic contrast to the 60% to 70% decrease in force previously demonstrated for unconditioned EDL muscles 3 days following this in situ LCP (11), the conditioned EDL muscles in the present study showed a decrease in force at 3 days of only 16% ± 4% for both age groups. After the final in situ evaluation, conditioned and contralateral unconditioned EDL muscles were dissected tendon-to-tendon and then were blotted dry on filter paper and weighed. Many previous investigations have identified that ~97% of the fibers in the EDL muscles of rats are fast type II (IIa, IIb, or IIx) fibers (20, 21), and no slow fibers were encountered or included in these experiments. Following the excision of the muscles, all rats were administered an overdose of pentobarbital sodium, and successful euthanasia was assured by opening of the thoracic cavity.

Muscle fiber preparation and attachment to force-position recording equipment. Bundles of fibers were dissected from each muscle and prepared for analysis of single permeabilized fibers. The preparation of permeabilized muscle fibers and attachment of the fibers to the force recording equipment has been described previously (6, 25, 26, 30, 31). The excised muscles were tied at approximately resting length to capillary tubes with silk suture, placed in a glycerol-based skinning solution, and stored at –20°C for up to 3 mo until required. On the day of an experiment, single fiber segments were carefully separated from the muscle bundle that was immersed in a petri dish filled with skinning solution. Small loops of 9-0 braided silk suture were tied to each end of an isolated fiber, and the fiber was then transferred to a small bath filled with relaxing solution [with a composition (in mM) of 7 EGTA, 20 imidazole, 5.4 MgCl2, 14.5 creatine phosphate (CrP), 4.74 ATP, 79 KCl, and 16 CaCl2, pCa 9.0]. One end of the fiber segment was tied directly to a fixed post attached to a force transducer (model 400A Cambridge Technology, Cambridge, MA), and the other end was tied directly to a post attached to the lever arm of a servomotor (model 300, Cambridge Technology). This method of fiber attachment has been shown to reduce fiber end compliance significantly (25, 26). A Polaroid photomicrograph (x400) was taken, and the length of 100 sarcomeres was measured with an ocular graticule. On the basis of this measurement, the length of the fiber segment (Lf) was set to provide a sarcomere length of ~2.6 µm, which is within the optimal range for maximum force production of single muscle fiber segments. Fiber width was measured using a stereomicroscope (Wild M3Z, Wild Heerbrugg) coupled with a high-power objective and camera system (Models MPS 51 S and MPS45, Wild Heerbrugg). Fiber cross-sectional area (CSA) was estimated assuming a circular fiber geometry with a subsequent adjustment to an ellipse by dividing by 1.7, based on previous direct measurements of width and depth of fibers (6, 26, 30). All displacements of the servomotor lever arm and force sampling of single fibers were controlled by microcomputer running ASYST software (Macmillan Software, New York, NY). Experiments were performed at 15°C.

Contractile activation and single LCP. Fibers were maximally activated by immersion in a bath containing activating solution with the following composition (in mM): 7 EGTA, 20 imidazole, 5.3 MgCl2, 14.5 CrP, 4.81 ATP, 64 KCl, and 7 CaCl2, pCa 4.5. To maintain structural stability, fibers were cycled between an isometric contraction and short periods of isovelocity shortening (2 Lf/s), followed by a rapid return to initial Lf (3). For the collection of experimental data, fibers were stretched between cycles. The maximum isometric force (Po) was measured at Lf immediately before each stretch. In each experiment, a fiber was maximally activated, and when force had plateaued at Po, the fiber was subjected to a single stretch of one of 5%, 10%, 20%, or 30% strains (% Lf) at a velocity of 0.5 Lf/s. Immediately following each single lengthening contraction, while the fiber was still activated maximally, isometric force was recorded after a steady state had been reached. The magnitude of injury was assessed by the force deficit [difference in maximum force (Po) before and after a lengthening contraction, expressed as a percentage of initial Po], which is the best quantitative measure of the totality of the injury (6, 25). Typical force traces of single permeabilized fiber segments before, during, and after lengthening contractions at 0.5 Lf/s have been presented previously (6, 8, 25).

The average force (mN) developed during a stretch was calculated by integrating the area under the force curve during the period of the stretch and dividing by the elapsed time (30). The values for Po and average force were normalized (kN/m2) on the basis of the estimated CSA of each fiber. Since some single permeabilized fiber segments can be damaged during removal from the bundles and attachment to the apparatus and subsequently generate abnormally low forces, experiments were performed only on fibers that developed forces of 100 kN/m2 or greater (25). Electron micrographs from a previous study revealed no structural damage to sarcomeres of permeabilized fibers compared with fibers from intact muscle before exposure to lengthening contractions (30). After each lengthening contraction, the work done to stretch a fiber was calculated from the product of the average force developed during the stretch and the displacement. Fiber mass was calculated from the product of Lf and the fiber segment CSA, assuming a density of 1 mg/mg3. Values for work done (J/kg) during the stretch were normalized by the mass of the fiber (kg).

Criteria for inclusion of data from single fibers in analyses. Since the magnitude of the force deficit is correlated highly (r = 0.93) with the force developed during the stretch, for both whole muscles (6) and single fibers (9), fibers that developed <100 kN/m2 force during maximum activation were eliminated from further consideration. These "weak" fibers constituted <10% of the fibers measured. Also not considered in calculations of force deficits were fibers classified as having undergone "breakage." Breakage of a fiber was noted if the fiber 1) broke into two parts, 2) showed visual evidence of tearing or disruption, or 3) displayed major inconsistencies in the development of force during or immediately after the lengthening contraction. For the majority of fibers, the force following the lengthening contraction reached a steady value that was maintained without decrement for many seconds to minutes. Furthermore, when fibers were relaxed and then reactivated, the force generated during the subsequent activation was not different from the steady poststretch force value. In some cases, the poststretch force did not remain steady, but slowly declined. In other cases, the force developed upon reactivation was inconsistent with the value recorded immediately poststretch. If the force level immediately following the stretch was not consistently maintained or if the force developed upon reactivation differed by >5% from the poststretch force value, the fiber was deemed to display "inconsistencies in the development of force." Most fibers that displayed such inconsistencies also showed visibly identifiable disruptions to the overall integrity of the fiber, although such disruptions could not always be verified visually. The percentage of breakages was calculated as the number of fibers that demonstrated breakage, as defined above, divided by the total number of fibers tested for a specific experimental group.

Determination of the number of sarcomeres in series (serial sarcomeres). Following training of rats with downhill running (27, 28), the conditioning of hamstring muscles of humans with lengthening contractions (4), and in several review articles (36, 37, 40), the decrease in the susceptibility of conditioned muscle fibers to contraction-induced injury has been attributed to an increase in the number of sarcomeres in series within the fiber. Consequently, in addition to an evaluation of force deficits following single stretches of permeabilized young- and old-unconditioned and young- and old-conditioned fibers, portions of the conditioned and unconditioned EDL muscles were prepared for analysis of serial sarcomere numbers. Muscles were fixed in 8% paraformaldehyde and, after fixation, digested in 30% nitric acid. Small fiber bundles were teased randomly (~15 per muscle) from each muscle, and fiber bundle length was measured using digital calipers. Sarcomere length was measured at three points along each fiber bundle using a digital imaging system coupled to a microscope. Sarcomere number for each bundle was estimated by dividing bundle length by average sarcomere length for the bundle. Sarcomere number was averaged across bundles to provide a representative value for each muscle.

Statistical analysis. Results are presented in Table 1 as means ± SE. Comparisons between the contractile parameters of single fibers from young and old rats following stretches at strains of 5%, 10%, and 20% were performed separately. The data for each strain were assessed using a one-way analysis of variance. A two-tailed t-test assuming nonequal variance was used to identify specific differences between groups when significance was detected. Serial sarcomere numbers from conditioned and contralateral unconditioned muscles were compared by paired t-tests. In all cases, results were considered significant if P < 0.05.


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  		Table 1. Comparison of contractile parameters of single fibers from extensor digitorum longus muscles from young and old rats during a single lengthening contraction at different strains

 

   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
A comparison of selected contractile properties of permeabilized single fibers from the EDL muscles of young and old rats showed no effect of age or conditioning on the force-generating capacity of single fibers or the work done on the fiber during stretch (Table 1). Electron micrographs of 1) a quiescent young-control fiber and 2) a young-control fiber fixed at resting length immediately after a 30% strain are shown in Fig. 1. Electron micrographs of single fibers demonstrated the nature of the sarcomere disruption that occurred during a severe lengthening contraction. Damage to the structure of both single sarcomeres and several sarcomeres adjacent to one another was apparent, but numerous intact sarcomeres surrounded the area of damage. Note also the stability of the z-lines throughout the fiber. The sarcomeric damage displayed in these micrographs represents the type of damage that resulted in the force deficits that we report.


Figure 1
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  		Fig. 1. Photomicrographs showing injury to individual sarcomeres in single fiber segments from extensor digitorum longus (EDL) muscles of 6-mo-old rats immediately following a lengthening contraction. A: control (uninjured) muscle fiber; magnification: x3,120. B: injured muscle fiber after a single lengthening contraction of 20% strain that produced a force deficit of 16%; magnification: x1,850.

 
After single stretches of maximally activated young- and old-control fibers, the magnitude of the force deficits increased with each increase in the magnitude of the strain (Fig. 2). Following a single stretch of 5% strain, the force deficits were not different for old- (2.3 ± 0.7%, n = 10) and young-control (2.5 ± 0.5%, n = 10) fibers, but following larger strains, the force deficits were greater for old- (10% strain: 9.0 ± 1.6%, n = 8 and 20% strain: 20.2 ± 1.2%, n = 14) compared with young-control (10% strain: 5.0 ± 0.8%, n = 11 and 20% strain: 14.9 ± 1.1%, n = 25) fibers. In contrast, when young- and old-control fibers were stretched with a 30% strain, the force deficits were not different (P = 0.122, two-tailed t-test; P < 0.06, one-tailed t-test), for fibers from old- (27.3 ± 1.7%, n = 9) compared with young-control fibers (23.4 ± 2.0%, n = 13).


Figure 2
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  		Fig. 2. Force deficit observed immediately following single stretches of varying strains of maximally activated single permeabilized fiber segments from the EDL muscles of young (solid symbols) and old (open symbols) rats. Fibers are from muscles of caged sedentary control rats (circles), muscles exposed to 6 wk of conditioning with a lengthening contraction protocol (inverted triangles), and contralateral unconditioned muscles (squares). All stretches were imposed at a velocity of 0.5 fiber length (Lf)/s. Strain (displacement) is expressed as a percentage of optimum Lf, and the force deficit observed after the stretch is expressed as a percentage of the isometric force developed just before the stretch. The number of fibers represented in each symbol is given in Table 1. *Significant differences in the force deficit between ages within the same experimental group. **Significant effect of conditioning within an age group.

 
Breakages for young-control fibers did not occur at the lower strains and were extremely rare overall. In contrast, breakages were frequent for old fibers and showed a rapid, more or less linear increase in occurrence with increasing strain to a frequency of 22% at a 20% strain and 45% of fibers demonstrating breakages at a 30% strain (Fig. 3). Consequently, the breakage rates were higher for the old- than for the young-control fibers at each strain, and the difference between the two age groups became much greater at the higher strains. The CSAs of old-control fibers were not different from those of young-control fibers with respect to either the mean values or the variability (Fig. 4). Furthermore, although the force deficits at a 20% strain were higher for old- than young-control fibers, the force deficits bore no relationship to CSA for either young-control or old-control fibers (Fig. 4).


Figure 3
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  		Fig. 3. Incidence of breakage in fibers from EDL muscles of young and old rats. The data are for a subset of the fibers that were activated and stretched and either remained intact or broke during stretch. Not every fiber listed from the subset was included in the final analysis if the criteria for inclusion were not met (e.g., low maximum isometric specific force; see METHODS for details). These data provide further insight into the increased susceptibility to damage during activation and stretch by fibers from old compared with young rats.

 

Figure 4
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  		Fig. 4. Individual fiber cross-sectional areas (CSA) and the force deficits following single stretches of 20% strain. Each symbol represents an individual fiber, and data are shown for all of the experimental groups. There was not a significant relationship (P = 0.43) between force deficit and fiber CSA.

 
Because of the substantial variability in the process and point during lengthening of fiber breakages, the force generated at the point of failure was not recorded routinely. Despite the omission of overall "failure forces," we did record peak stresses during stretches of 20% strain for unconditioned and conditioned fibers and compared the fibers that were ultimately eliminated due to inconsistencies in force development with fibers that remained intact and produced steady forces. Although the maximum isometric specific force (Po) for fibers eliminated due to an inconsistent force response following the stretch was not different from the Po for intact fibers that produced steady forces, peak stresses during the stretch were 10% lower (P < 0.05) for fibers that "failed" (289 ± 15 kN/m2) than for fibers that remained intact (321 ± 7 kN/m2). These results are consistent with the concept that fibers may have been "failing" during the stretch, and, moreover, since the number of fibers that failed was greater for old compared with "adult" fibers, failure stress appears to be decreasing with age and increased by conditioning.

Although a highly significant protection from contraction-induced injury was observed at the whole muscle level for the conditioned EDL muscles of both young and old rats, the force deficits for the young-conditioned single fibers were not different from those for the young-unconditioned fibers. In contrast, conditioning had a dramatic effect on the force deficits of the old fibers, with the force deficit reduced from 20.2 ± 1.2% for old-unconditioned fibers to 15.0 ± 1.1% for old-conditioned fibers. As a result of the decrease of 25% in the force deficit of the old-conditioned fibers, the force deficits of the young- and old-conditioned fibers were not different. In addition to the decrease in the force deficit for old-conditioned compared with old-unconditioned fibers, following the single 20% strains, no breakages were observed among the conditioned fibers from either young or old animals. Despite the 25% decrease in the force deficit and the near complete elimination of breakages observed following 6 wk of conditioning, no differences were observed among the groups for the number of sarcomeres in series along the length of fibers (unconditioned fibers: 4,708 ± 131 sarcomeres, n = 6 muscles; conditioned fibers: 4,445 ± 93 sarcomeres, n = 6 muscles). In addition, the lack of any relationship between force deficit and CSA was maintained following the 6 wk of conditioning with the LCP (Fig. 4). Thus, the decrease in response to the conditioning in force deficit for old fibers was not explained by larger CSAs of the conditioned fibers. Furthermore, no difference was observed in either age group between the CSAs of the fibers that underwent breakage (2,572 ± 160 µm2) compared with those that did not (2,210 ± 92 µm2).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In our current study, as in a previous study (6), force deficits were twofold greater following single 10% stretches of maximally activated permeabilized single fibers from old compared with young rats. In both sets of data, a considerable reduction was observed in the difference in force deficits between young and old fibers during a 20% strain. In the present study, following a 30% stretch, the force deficits were not different between age groups. Thus, a consistent finding of the two studies was a substantial decrease in the magnitude of the difference between the force deficits experienced by young-control and old-control fibers as strains were increased. Whole, fast-fibered, EDL muscles of mice displayed a linear increase in the force deficit as the strain was increased from 20% to 60% (10), and young-control EDL fibers also displayed an essentially linear increase in the force deficit for stretches of from 5% to 30% strain. In contrast, old-control fibers displayed a curvilinear relationship between force deficit and strain. Compared with the force deficits predicted by the line extrapolated from the force deficits at strains of 5% and 10%, the force deficits measured at a strain of 20% were 20% lower and at a 30% strain were 37% lower.

No evidence of fiber breakage or tearing was observed when whole EDL, or soleus, muscles of young mice were tested in vitro with single lengthening contractions, even up to strains of 60% relative to fiber length (10). In the present study, breakages had little or no impact on the force deficits of the young-control fibers since breakages were almost nonexistent until a 30% strain was imposed, and even then breakages were few. The strong linear relationships between force deficit and the strain observed for whole muscles (6, 9, 10) and for young fibers (present study; Refs. 6 and 30) support a high correlation between these two variables in the absence of outright breakages. In contrast, the curvilinear relationship between force deficit and strain for old-control fibers, with a flattening of the force deficits over the same range of strain, 10% to 20% of Lf, over which the number of breakages doubled from ~20% to ~40%, strongly suggests a substantial influence of the number of fiber breakages on the relationship between force deficit and strain. Furthermore, the close association between the increasingly large number of breakages incurred by the old-control fibers at strains of 20% and 30% and the deviation from linearity for the force deficit-strain relationship supports the conclusion that fiber breakages explain the apparent lessening of the difference in magnitude of force deficit for old- and young- fibers at higher strains. Consequently, the force deficits of single fibers can only be determined reliably and compared at strains where the breakage rates for both ages are relatively low. Trappe and colleagues (45, 46) have similarly reported high breakage rates for fast type IIa fibers from vastus lateralis muscles of older women both before and after progressive resistance training with shortening contractions.

Despite the decreased structural integrity of fibers with aging, reflected in the high incidence of fiber breakage and the large force deficits observed for old-control fibers, the loss of structural integrity apparent during lengthening contractions was not associated with lower specific forces measured during isometric contractions. The differences between these two types of contractions indicate that, although the capacity of the contractile apparatus to generate isometric force is largely preserved with age, the vulnerability of the fibers to damage induced by stretch is not. For stretches of 20% and 30% strain, the old fibers that were sampled and tested successfully appear to represent a particularly robust subpopulation of fibers that were able to withstand these strains. The uncoupling observed in the present study between the isometric contractile function and the fragility of permeabilized single fibers during maximally activated stretches is extremely interesting and warrants further investigation.

Although when normalized by tissue mass, the work required to stretch permeabilized EDL muscle fibers and whole EDL muscles through a 20% strain was not different, 72 J/kg and 60 J/kg, respectively, the resultant force deficit for the single fibers was eightfold greater. The greater magnitude of the force deficit for single permeabilized fibers compared with intact muscles is not explained by effects of the fiber permeabilization and isolation procedures per se. As indicated in METHODS, we used very strict selection criteria to exclude fibers that were damaged through isolation or mounting to the force recording apparatus. Rather than indicative of effects of the process used to obtain single fibers, the greater force deficits of single fibers compared with whole muscles support the critical role that neighboring fibers play in the protection from severe contraction-induced injury, as well as the protective effects normally conferred by an intact sarcolemma, the dystrophin-associated glycoprotein complex, and associated cytoskeletal proteins (15, 26, 35, 44).

Following single lengthening contractions of old- compared with young-control fibers, the twofold greater force deficits at a 10% strain support the concept of an age-related increase in the susceptibility of fibers to mechanical damage. In addition, the much higher breakage rates at each strain indicated an increase in the number of fibers at risk of being severely injured at each strain. Furthermore, while certainly not definitive, collectively our findings support the hypothesis that the single fibers that break are fibers that would have had large force deficits had they been tested at lower strains. These observations are consistent with the widespread observation that, for humans (34), rats (6, 30, 31, 38), and mice (6, 52), fibers in, or from, skeletal muscles of old animals are much more susceptible to contraction-induced injury. A wide range of inventive studies involving humans stepping down (38), performing negative work pedaling (18), executing drop and rebound jumps (21), with stretches of maximally activated hamstring (4) and bicep muscles (38), as well as stretches of maximally activated muscles of mice (9, 49), or permeabilized single fibers from rats (6, 30, 31) have attempted to identify the mechanism responsible for the immediate injury to fibers through histological, histochemical, or electron microscopic assessments. The overwhelming evidence, particularly the evidence based on electron microscopic sections of single fibers at high power, is that the immediate injury is to individual sarcomeres, or small clusters of sarcomeres (9, 13, 31, 38, 39). Whether in old animals muscle fibers in vivo experience additional disruptions related to the fragility observed for permeabilized fibers in vitro has not been addressed experimentally, but this issue could represent an additional risk for older persons who perform physical activities that involve lengthening contractions.

In a previous study of adult and old mice, 6 wk of conditioning of the dorsiflexor muscles with an in vivo LCP had a dramatic effect on the susceptibility of the whole dorsiflexor muscle group to contraction-induced injury (8). Specifically, the conditioned EDL muscles had a force deficit 3 days after a LCP of 8 ± 2%, compared with 25 ± 2% for unconditioned EDL muscles, with no differences between adult and old age groups (8). Despite dramatically reduced force deficits for whole EDL muscles in the present study following a similar 6-wk program of conditioning with the LCP, no effect on the susceptibility to injury was observed for single fibers of young-conditioned compared with young-unconditioned muscles. In contrast, the 6 wk of conditioning completely eliminated both the high breakage rate and any difference in the magnitude of the force deficits between old-conditioned and young-conditioned fibers. For the initial injury caused by lengthening contractions, the prevailing hypothesis is that weaker sarcomeres in series with stronger sarcomeres are stretched excessively and damaged (9, 17, 31, 36–38). Heterogeneities in sarcomere strength may arise from random variations in resting sarcomere lengths or intrinsic differences in the relative strengths of different sarcomeres (36), and slower protein turnover in muscles of old compared with young animals (43) may result in a larger population of "older" and weaker sarcomeres. Whether sarcomeres on the descending limb of the length-force curve are pulled beyond overlap of thick and thin filaments (36), or if injury is intrinsic to single sarcomeres (30) has not been resolved, but damage to and degeneration of weak sarcomeres followed by regeneration of stronger sarcomeres is a potential mechanism for the increased resistance to stretch-induced damage of old-conditioned fibers. This mechanism is supported by the observation that newly regenerated fibers are clearly more resistant to contraction-induced injury in both young and old animals (11). Although the replacement of weak sarcomeres with stronger sarcomeres is expected to occur in both adult and old animals, we propose that this particular mechanism of protection has a more discernible effect in muscles of old animals that are hypothesized to have greater numbers of weak sarcomeres in the unconditioned fibers.

The lack of protection from initial mechanical injury observed in single fibers from conditioned muscles of adult rats indicates that additional protective mechanisms underlie the reduction in the magnitude of injury observed for whole muscles. Protective mechanisms that target the secondary injury as opposed to the initial mechanical stability of the sarcomeres are also invoked in both adult and old animals (20, 23). The effectiveness of the conditioning in the present study was validated in vivo as a reduction in the magnitude of injury observed 3 days following a protocol of lengthening contractions. This is a time point when numerous factors in addition to sarcomere damage, in particular inflammation and the release of free radicals and proteases, are contributing to the severity of the injury. Exercise conditioning programs induce adaptations that reduce the magnitude of this secondary injury even in the face of similar levels of initial injury (19, 20, 23).

In summary, our findings highlight unequivocally the greater susceptibility of old muscle fibers to contraction-induced injury, both in terms of the force deficits and in the frequency of fiber breakages. Despite the association of high fiber breakage rates and large force deficits with aging in cage-sedentary rats, these detrimental characteristics can be reversed by a conditioning program that involved repeated bouts of lengthening contractions. Furthermore, on the basis of the high breakage rates for old-control fibers, the study provides important insights regarding the extent to which contraction-induced damage can be studied reliably using permeabilized single fibers. The only valid comparison of force deficits between old-control and young-control EDL fibers was at 10% strain, where the difference was twofold. Almost every activity of daily living involves lengthening contractions. The majority of lengthening contractions do not induce injury, but the existence of a significant population of highly stretch-susceptible fibers in muscles of old animals place these muscles at a much greater risk. Despite their high susceptibility to injury (6, 30, 31, 29, 38, 52), muscles in old animals are capable of undergoing adaptations that result in protection from injury (5, 32, 38, 39). The findings from the present study confirm that conditioning programs of repeated lengthening contractions can be effective in protecting muscle fibers from contraction-induced damage and that the mechanism for adaptation is within the sarcomeres and not from the addition of sarcomeres. The findings raise critical questions regarding the advantages, as well as the potential risks involved in using lengthening conditioning programs for the elderly.


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This work was supported by National Institutes of Health Grant AG-13283 to the Contractility Core of the Nathan Shock Center.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. S. Lynch, Basic and Clinical Myology Laboratory, Dept. of Physiology, The Univ. of Melbourne, Victoria, 3010, Australia (e-mail: gsl{at}unimelb.edu.au)

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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