Lynn, R., and D. L. Morgan. Decline running produces more sarcomeres in rat vastus intermedius muscle fibers than does incline running. J. Appl. Physiol. 77(3): ...
Decline running produces more sarcomeres in rat vastus intermedius muscle fibers than does incline running R. LYNN AND D. L. MORGAN Department of Electrical and Computer Clayton, Victoria 3168, Australia
Systems
Lynn, R., and D. L. Morgan. Decline running produces more sarcomeres in rat vastus intermedius muscle fibers than does incline running. J. Appl. Physiol. 77(3): 1439-1444, 1994.-Unaccustomed eccentric exercise, in which a muscle is lengthened while generating tension, is well known to cause injury and pain. A rapid training effect has been demonstrated in a number of eccentric exercises. The mechanism for both the damage and the training has been unknown. Morgan proposed that the damage is caused by sarcomere length instabilities during operation on the descending limb of the sarcomere length-tension curve and that the training effect is an increase in the number of sarcomeres connected in series in a muscle fiber, thus avoiding the descending limb (Biophys. J. 57: 209221, 1990). We tested this proposal by exercising rats on a treadmill set at either an incline or a decline of 16”, an exercise that has previously been shown to cause damage in untrained rats and a training effect. The vastus intermedius muscles were fixed and were digested in acid, and the fiber and sarcomere lengths of representative fibers were measured. From these measurements, the mean number of sarcomeres per fiber was found for the different training regimes. A clear and repeatable difference was found, supporting Morgan’s prediction of more sarcomeres after decline running, although with some differences in response that depended on the age of the rats. muscle mechanics; cle plasticity
eccentric
exercise; muscle adaptation;
mus-
for many years that exercises that involve stretch of active muscle tend to produce “delayed-onset muscle soreness,” in which the muscles involved show soreness and tenderness for up to 1 wk, peaking l-2 days after the exercise (4,14). It has become clear that such soreness is associated with damage to muscle fibers (1,3,6,7,9,11,16). The damage is thought to involve loss of calcium homeostasis, probably by release of calcium from the sarcoplasmic reticulum, but the mechanism initiating the damage has not been clear (2,3, 7). Indeed, most of the plausible suggestions are not supported by evidence. Two of the distinctive features of eccentric exercise damage are that even a single bout of training provides significant protection against damage from subsequent such exercise and that is there is a very substantial and rapid training effect for this type of exercise (6, 8, 17). The physiological mechanism underlying this rapid training effect of eccentric exercise has been unknown, and even plausible theories have been lacking. Morgan (15) considered the lengthening of muscle with sarcomeres on the descending limb of the sarcomere length-tension curve, that is, sarcomeres with a tensiongenerating capacity that decreases with increasing length. He came to the conclusion that lengthening of an active muscle fiber or myofibril occurs more nearly by “popping” of sarcomeres (or half-sarcomeres) one at a IT HAS BEEN KNOWN
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time in order from the weakest to the strongest than by near-uniform lengthening of sarcomeres. The term popping was chosen to emphasize that the lengthening was rapid and uncontrollable because of an instantaneous instability produced by the decreasing length-tension curve and the flat force-velocity curve beyond the yield point observed in the lengthening part of the curve. The theory predicted that the popped sarcomeres would be stretched to the point where the total of active and passive tension equaled the total tension at the initial length. At least for frog muscle, this is beyond the region of overlap between thick and thin filaments. This theory was able to explain many otherwise puzzling observations. Morgan (15) further suggested a mechanism for the damage of eccentric contraction with the following steps. In a small proportion of the overstretched sarcomeres, the filaments may fail to resume their interdigitating pattern on relaxation, leaving a sarcomere with less filament overlap and thus fewer active bridges than before. On the next stretch, this sarcomere will generate less tension and will thus pop early in the contraction, straining neighboring sarcomeres. The process is then repeated on the next eccentric contraction, leading to the creation of a patch of poorly interdigitating sarcomeres that are overstretched during activation. This irregularity will strain the transverse tubules and the sarcoplasmic reticulum. After a large number of such muscle stretches, these patches of overextended sarcomeres may lead to damage of membranes, release of calcium from either the sarcoplasmic reticulum or the extracellular space, and so to the fiber damage reported. This suggestion is consistent with electron-microscopic observations of muscle immediately after eccentric exercise, which show small regions of overextended sarcomeres, ranging from a single sarcomere in a single myofibril to a few sarcomeres extending across a significant part of a fiber (3, 16, 18). A corollary of this proposed mechanism was the suggestion that the training effect of eccentric exercise may come about by an increase in the number of sarcomeres in series in muscle fibers such that the sarcomeres stay on the ascending limb of their length-tension curves over the range of muscle lengths used in the exercise and thus avoid the sarcomere length instability and the consequent damage (15). The experiments reported here set out to test this suggestion by estimating the number of sarcomeres in representative fibers from the vastus intermedius muscle of rats exercised by either incline or decline running on a treadmill. Armstrong et al. (3) and Schwane and Armstrong (17) have previously shown that this exercise produces damage in untrained rats and that the training protocol used here produces rats that do not suffer dam-
0 1994 the American Physiological Society
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1440
TRAINING
TABLE Expt
OF
ECCENTRIC
EXERCISE
1. Details of the six experiments performed
No.
No. of Animals in Group
Training
1
Decline Incline Decline Incline Decline Level Sedentary Decline Incline Sedentary Decline Incline Sedentary Incline Incline Incline Sedentary
2* 3
5
6
* Both
EFFECT
groups
were given
Training Duration,
wk
Average Age, days
Sex
101
Female
140
Male
350
Male
300
Male
1 1
100
Male
1 2 3
106
Male
1 2 5
4 1 wk of incline
running
before
Body
Wt,
g
205 206 345 348 478 483 500 449 462 450 351 349 360 381 365 352 330
test week.
age from a prolonged bout of downhill running. Hence, these experiments deal with trained muscles and not damaged muscles. METHODS For each of six experiments, groups of Long-Evans rats of uniform age, weight, and sex (Table 1) were exercised on a small-animal treadmill according to a slight modification of the protocol of Schwane and Armstrong (17). Exercise consisted of alternations of 5 min of running at 14 m/min and 1.5 min of rest. The exercise period was 15 min on the 1st day and increased to 30 min on the 4th and subsequent days. This gradual introduction to the exercise avoided the need to use electrical stimulation to induce the rats to run. The slopes used were 16O incline and 16’ decline, and in experiment 3 the incline was level. Three days after the last training run, the rats were killed with carbon dioxide. Each vastus intermedius muscle, still attached to the femur and tibialis bone, was removed. The angle of the knee joint was set to m-60”, and the muscles were fixed in buffered formaldehyde (pH 6.9) and digested in 30% nitric acid (12). The vastus intermedius muscle was chosen because Schwane and Armstrong had shown that extensive damage to it during decline running in untrained rats was substantially reduced by previous decline training as used here. Muscles were processed in random order with the operator unaware of the group to which the muscle belonged. The fibers were dispersed using an ultrasonic cleaning bath and were serially diluted until an aliquot containing a manageable number of single fibers was obtained. Most of the intact fibers in this sample were then analyzed so that operator selection of fibers was minimized. Fibers were placed one at a time on a concavity slide and were examined under a binocular microscope with a television camera att ‘ache d. Fibers were examined carefully at both ends to ensure that they were not broken. The suitable fibers were then photographed by capturing an image from the camera onto a computer [Macintosh IIfx with 21-in. screen (Apple Computer, Cupertino, CA) using a Quick-Image frame capture board]. The lengths of fibers were estimated by approximating the fiber image with a series of straight lines using an image-processing application (IPLab, Signal Analytics, Vienna, VA). Sarcomere length was measured by obtaining a diffraction pattern from the center of the fiber with use of a helium-neon laser. The estimated sarcomere count was found by dividing the fiber length by the sarcomere length.
Note that both fiber length and sarcomere length depend on the joint angle but that sarcomere count is the fundamental structural parameter independent of fixation conditions. For this reason attention was focused on the sarcomere counts and not the sarcomere or fiber lengths. To provide some information on changes in tendon length and hence fiber length at a given joint angle, extra care was taken in experiment 4 to fix all the muscles at the same joint angle. Several tests were done to estimate the accuracy of the technique. The first was designed to assess the error involved in only measuring the sarcomere length at one point. In 18 fibers from an incline-trained animal and in 18 fibers from a declinetrained animal, the sarcomere length was measured at 0.5mm spacings along the fiber. For each fiber, the average of these readings was compared with the sarcomere length at the center point, and the mean magnitude of the difference was found to be 1.55% for fibers from the inclined-trained rats and 0.8% for fibers from the decline-trained rats. In the second test, each of 24 fibers was subjected to the complete measurement process three times, and the range was found as the difference between the largest and smallest of the three values. The mean value of this range was 2.92%. Because our conclusions were based on differences in the mean estimated sarcomere count between pools, the third test involved finding the mean of a pool of 24 fibers five times over. These measurements were done in random order, and the difference between the largest and smallest values obtained for the mean of the 24 fibers was 0.62%. The use of laser diffraction to measure sarcomere length relies on uniformity of sarcomere lengths. Although microscopy of muscle immediately after a single burst of eccentric exercise shows considerable sarcomere inhomogeneity, all the evidence suggests that the repair processes should have removed inhomogeneity in these muscles, which were fixed 7 days after the initial training bout and 3 days after the last one. Schwane and Armstrong (17) showed that biochemical markers at the corresponding time of a nearly identical protocol returned to normal, and Friden et al. (9) state that “fiber structure and muscle strength was then essentially normal 6 days after exercise.” Our experiments did not show any difference between the homogeneity of fibers from incline- and decline-exercised rats as indicated by laser diffraction. These tests convinced us that most of the observed width of the histograms of estimated sarcomere number was due to genuine variation between fibers within the muscles and not to measurement uncertainties. This made the sampling process very
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1 i n e
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Declinemean= 3526 sem=34
Sedentary----. mean=3259
Number
of sarcomeres
important to the result, leading to the serial dilution technique described above to ensure random sampling of fibers from each muscle. Similarly, we adopted the “blind” processing procedure so that there was no opportunity for inadvertent bias in the limited choosing of fibers that was necessary. Statistical testing used Stat-View (Abacus Concepts, Berkeley, CA). Igor (Wavemetrics, Lake Oswego, OR) was used for data processing, including construction of histograms of sarcomere counts and fitting of gaussian curves. Experiments were conducted in accord with the Declaration of Helsinki and were authorized by the Monash University Animal Ethics Committee approvals 89/11O and 92/O&3. RESULTS
In the basic experiment, four rats were trained on an incline of 16” and four rats were trained on a decline of the same magnitude for 1 wk. The two groups were chosen to match sex, age, and body weight, as detailed in Table 1. Figure 1 shows the histograms from a typical experiment (experiment 4; Table 1). The average number of sarcomeres after incline training was 89% of that after decline training. This difference was significant at the P < 0.0001 level by unpaired t test, one-factor analyis of variance, and Kruskal- Wallis nonparametric analysis. The sedentary rats had a sarcomere count equal to 92.4% of that for decline-trained rats, which is not significantly different from the incline value at the P < 0.05 level. This finding shows that the difference between incline and decline training in these rats was mainly an increase due to decline running rather than a decrease due to incline running, in accordance with Morgan’s prediction (15). In this experiment, the angles of fixation were carefully controlled and fiber lengths at a joint angle of 60” were compared. The average fiber length for the fibers measured from the incline-exercised rats was 98.8% of that of decline-exercised rats and was not significantly different. Five of the six experiments performed according to the
protocol described and listed in Table 1 included a comparison of decline training with either incline training or level running. The results of these experiments are summarized in Fig. 2. For all five experiments, the incline count was significantly less than the decline count; that is, the ratio of the incline-trained to decline-trained sarcomere count was significantly lessthan unity. However, the magnitude of the difference and the relative value of sedentary control rats varied somewhat with the age of the rats. Experiment 1 (lOl-day-old rats) resulted in an incline count of 94.9% of decline. In experiment 5 loo-day-old rats and including a sedentary group, the mean sarcomere count for 240 fibers from incline-exercised rats was 3,266 t 26,93.3% of the count of 3,501 t 27 (n = 240) for decline-exercised rats (P < 0.0001 by t test). However, the sedentary rat fibers (n = 240) had a mean count of 3,421 t 23,97.7% of that of the decline-exercised animals, which is significant from the incline (P < 0.0001 by t test) and decline training results (P = 0.022). Thus the young sedentary rats had counts closer to the decline-trained than the incline-trained rats, suggesting that both slopes produced some training effects but in opposite directions. Experiment 2 used 14O-day-old rats that had all been trained by incline running fo r 1 wk before the week of incline or decline running. That is, four rats were trained on the incline for 2 wk, whereas the other four were trained by incline running for 1 wk and by decline running for the 2nd wk. This procedure was intended to minimize any effects of variability between the animals due to their exercise levels before coming into our care, although subsequent experiments suggested that it was not necessary. The difference here was very clear with incline being 89.7% of decline, which again was significant (P < 0.0001). A single sedentary rat gave sarcomere counts near that of the incline-trained group (data not shown) 9supporting the finding that you nger animals decrease their sarcomere counts with incline training com-
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EFFECT
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ECCENTRIC
EXERCISE
1.00
l
o
Incline Sedentary
level I
1
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I
.
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i
1 I
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0.85 I 0
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I 200
I 100 100
Average
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I 300
FIG. 2. Summary of experiments. Mean sarcomere counts for incline-trained and control groups for each experiment are expressed as fraction of mean sarcomere count for declinetrained runners of same experiment plotted against age of rats. Error bars, SE of ratios (found from SE of means of groups as described in Ref. 13). In 1 experiment (level), there was no incline group but a group trained on the level. All incline and level ratios are significantly
