Histochemical and physiological correlates of training- and detraining-induced changes in the recovery from a fatigue test.

Key Words: Energy expenditure, Exercise, strengthening, Fatigue, Functional training and activities, Pulmonary, tests and measurements

Tests of local muscle endurance able to detect changes induced by exercise training or detraining would be of great benefit in rehabilitation settings. Local muscle endurance is influenced by muscle fiber type composition.[1-7] Some reports have suggested that the rate of force decline from repeated maximum isometric or isokinetic muscle contractions varies as a function of the percentage of slow-twitch (type I) or fast-twitch (type II) muscle fibers within the active musculature.[1,2,4] Thorstensson and Karlsson[2] found that the decline in maximal force after 50 repeated isokinetic contractions at 180[degrees]/s, expressed as a percentage of initial torque values, was highly correlated (r = .86) to the percentage of fast-twitch fibers in the vastus lateralis muscles of male college students. Although these reports clearly demonstrate a relationship between muscle fiber distributions and the decline in force (torque) over repeated contractions, it is unclear which muscle fiber type plays the dominant role. It appears the relationship is very much dependent on a number of factors, including the type of muscle contraction tested, the length of the fatiguing bout, the absolute torque levels of subjects studied, and possibly differences in muscle fiber type distributions in the subjects.

Although the percentage of decline in peak torque at the end of a standard isokinetic fatigue test (ie, what is often called the "fatigue index"[2]) may be related to the percentage of fast-twitch muscle fibers, the percentage of decline in torque from initial values may not reflect the adaptive changes in skeletal muscles resulting from intense endurance exercise training or detraining. Because the percentage of total fast-twitch and slow-twitch muscle fibers is not appreciably changed as a result of endurance exercise training or detraining,[8,9] the percentage of decline in torque at the end of a fatigue test may not reflect training-or detraining-induced changes, especially if a large determinant in the development of fatigue is the percentage of type I fibers. The recovery of peak torque from a brief bout of fatiguing exercise, however, could be influenced by the relative proportions of type IIa and type IIb muscle fibers and the changes in the relative percentages of these skeletal muscle fibers in individuals undergoing endurance exercise training or detraining. Furthermore, it seemed possible that the recovery of muscle peak torque following a fatiguing bout of isokinetic exercise could be influenced by maximal aerobic exercise capacity ([Vo.sub.2]max).

The major purpose of this study was to test the hypothesis that the recovery of muscle peak torque is a more sensitive measure of endurance exercise-induced adaptations in skeletal muscle than the percentage of decline in torque during a fatigue test. A secondary purpose was to obtain information regarding the physiological factors associated with changes in recovery of peak torque induced by endurance training. To this end, we assessed the relationships between the changes in quadriceps femoris muscle performance during recovery from an isokinetic fatigue test and (1) the changes in quadriceps femoris type II muscle fiber distributions following 12 weeks of intense endurance training and 12 weeks of detraining (ie, cessation of intense endurance training) and (2) the changes in maximal oxygen uptake capacity ([Vo.sub.2]max).

Method

Subjects

Three groups of nondisabled subjects were studied to determine whether the recovery or muscle peak torque after a standard fatigue test was related to different physiological and histochemical variables. Four subjects (2 male, 2 female) underwent endurance exercise training for 12 weeks on a bicycle ergometer. These subjects had not engaged in regular endurance exercise for at least 6 months prior to the study. Six endurance-trained subjects (5 male, 1 female) also agreed to discontinue all endurance exercise training (ie, detrain) for 12 weeks. The subjects who underwent detraining have been described previously[9]; we report the results of 6 of the original 7 subjects, as 1 subject did not complete 12 weeks of detraining.

An additional 11 subjects (8 male, 3 female) volunteered to perform a single quadriceps femoris muscle fatigue test with recovery and a graded treadmill or cycle ergometer test to determine [Vo.sub.2]max. These subjects did not exercise train or detrain or have muscle biopsies, but were included to study the relationship between a local measure of quadriceps femoris muscle endurance (recovery of peak torque) and a central measure of endurance capacity ([Vo.sub.2]max). Every effort was made to solicit the participation of subjects who engaged in a range of regular physical activity and who possessed a range of maximal endurance capacities. The physical characteristics of all three groups of subjects are presented in Table 1. All subjects agreed to participate and signed an informed consent form outlining the procedures.

[TABULAR DATA 1 OMITTED]

Procedure

Muscle biopsies and tissue preparations. Prior to and immediately following the 12 weeks of endurance exercise training or detraining, subjects underwent a muscle biopsy of the vastus lateralis or gastrocnemius muscle to determine the muscle fiber type composition. All four subjects who underwent exercise training had muscle biopsies of the vastus lateralis muscle and trained on the bicycle ergometer. Of the six subjects who underwent detraining, the three who were cyclists had biopsies of the vastus lateralis muscle and the three who were distance runners had biopsies of the lateral head of the gastrocnemius muscle. The lateral gastrocnemius muscle was selected for biopsy in the three distance runners because we assumed this leg muscle was more likely to demonstrate loss of adaptive changes associated with the cessation of running.[9] Our assumption was that the lateral gastrocnemius and quadriceps femoris muscles would demonstrate the same relative adaptive changes from detraining.

Using the needle biopsy technique described by Bergstrom,[10] muscle samples were obtained from the same area of the muscle before and after training or detraining. The muscle specimen obtained was oriented under a dissecting microscope, placed in OCT[R](*) (ornithine carbamoyltransferase), and then frozen in isopentane chilled in liquid nitrogen. All muscle samples were stored at less than -70[degrees]C until histochemical analysis. Several (approximately 12-20) transverse serial muscle sections (12-14 [mu]m in thickness) were made using an IEC Minot Custom Microtome([dagger]) maintained at a constant temperature of -20[degrees]C. Unfixed frozen cryostat sections were thaw-mounted onto coverslips for staining. Sections were stained for myosin adenosine triphosphatase (ATPase) after preincubation at pH 4.6 to 4.7 to determine the percentages of type I, type IIa, and type IIb fibers. Typically, one of the ATPase-stained muscle sections was used to determine the percentage of each fiber type. Classification of muscle fiber types was based on the differences in staining intensity and pH sensitivity of the myosin ATPase.[11] Alternate muscle sections were stained with reduced nicotinamide adenine dinucleotidetetrazolium reductase (NADH-TR) and ATPase after preincubation at pH 4.3 and 10.3 to verify classification of muscle fibers. All muscle samples from those subjects who underwent training or detraining were stained in the same glass container to control for differences in staining intensity.

Fatigue test and recovery. All subjects performed a 1-minute fatigue test of the quadriceps femoris muscle. This test consisted of 50 maximal isokinetic knee extensions at 180[degrees]/s on a Cybex[R] II isokinetic dynamometer, ([double dagger]) as previously described.[2] Each subject was seated with the hips between 75 and 90 degrees of flexion. The axis of rotation of the Cybex[R] II was aligned with the anatomical axis of the subject's knee. The Cybex[R] II lever arm was attached to the subject's leg at a distance approximately 70% of the length from the medial knee joint line to the medial malleolus to allow for a standardized and comfortable site. The subject's thigh was stabilized with a canvas strap fixed to the table. Subjects were asked to maximally extend their leg through a complete range of 90 degrees and were paced at a rate of 50 extensions per minute by use of a metronome. The subjects were instructed to allow the extended limb to passively return to full flexion prior to the next contraction. The torque generated during knee flexion was monitored continuously and remained small throughout the test's duration.

To assess the recovery of peak torque from this bout of fatiguing exercise, each subject performed a single maximal knee extension every 30 seconds for 3 minutes of recovery. The percentage of decline from initial peak torque (at the start of the 1-minute test) was calculated from the mean peak torques of the 46th through 50th knee extensions and at every 30-second interval for the 3 minutes of recovery. The initial peak torque was taken as the average of the first three knee extensions performed at the start of the test. Peak torque was obtained from the dual-channel strip chart recorder using a damp setting of 2, which remained constant for all tests.

Corrections for the torque required to lift the weight of subject's limb and the dynamometer's lever arm against gravity were not added to the obtained peak torques, because the differences in gravity-corrected and gravity-uncorrected values have been reported to be small (ie, 2.4%).[12] We assumed that small differences would not jeopardize our findings. Calibration of the Cybex[R] II dynamometer was performed with known loads at the testing velocity prior to each test using a damp setting of 2.

The subjects who underwent training and detraining each underwent two fatigue tests. The first test preceded the 12 weeks of training or detraining, and the second test immediately followed the 12-week program. The second fatigue test was performed within 48 hours of the exercise test to determine [Vo.sub.2]max.

Determination of [Vo.sub.2]max description of training protocol

All subjects who trained or detrained had a graded treadmill or cycle ergometer test prior to and immediately after the 12-week period. For the training subjects, exercise tests were repeated within 48 hours following their last training bout after the 12 weeks. Those subjects who underwent a treadmill test were evaluated while running up a grade on the treadmill. Work rate was increased every 2 minutes until the subject was unable to continue. Those subjects tested on the cycle ergometer had the work rate increased every 2 minutes by 150 kpm until they were unable to continue or maintain a pedal cadence of 60 rpm. Criteria for obtaining [Vo.sub.2]max was a leveling off of oxygen uptake with an increment in work rate and respiratory exchange ratios (RERs greater than or equal to 1.15. Expired gases were collected in neoprene meteorological balloons every 30 seconds for the last 3 to 4 minutes of each test. Expired gases were analyzed for fractions of oxygen and carbon dioxade with a mass spectrometer (Perkin-Elmer MGA 1100([section])), which was calibrated by gases analyzed by the Scholander technique. Expired volumes were measured with a Tissot chain-compensated spirometer.([parallel])

Training consisted of 12 weeks of endurance exercise on a cycle ergometer, 6 days per week for a duration of 45 to 60 minutes per day, at an intensity eliciting 70% to 90% of the subject's [Vo.sub.2]max. Subjects exercised the majority of time at 70% to 80% of [Vo.sub.2]max during the initial 3 weeks, at 80% to 85% of [Vo.sub.2]max during weeks 4 through 6, and at 85% to 90% of [Vo.sub.2]max during weeks 7 through 12. Work rates were adjusted weekly based on monitoring of heart rate-oxygen consumption relationships during an exercise session. During the last 6 weeks, subjects performed two to three sessions of interval training per week. A typical interval training session consisted of four or five 5-minute bouts of exercise at intensities eliciting 90% to 95% of [Vo.sub.2]max. All subjects tolerated the exercise sessions well.

The subjects who underwent detraining were instructed to limit their physical activity to the minimal level required by their sedentary jobs. Typically, these subjects walked less than 500 m per day at a slow pace and were instructed to refrain from stair climbing when possible. Except for the exercise testing at periodic intervals as previously described,[9] these subjects did not perform any regular physical activity.

Data Analysis

The changes in muscle fiber type distributions as a result of the 12-week training and detraining program were analyzed using the chi-square statistic. This statistic was used because fiber type distributions are frequency counts. Comparisons were made between the percentages of type I and II fibers and the percentages of type IIa and IIb fibers. The percentage of each muscle fiber type was derived from an average of 483 total muscle fibers counted in each muscle biopsy (Tabs. 1, 2).

[TABULAR DATA 2 OMITTED]

The percentage of reduction in torque at every 30-second interval throughout the 3-minute recovery period was analyzed using Student's t tests for paired observations. Bonferroni's correction was used to protect against a type I error using multiple t tests.[13] Pearson Product-Moment Correlation Coefficients were computed to determine the relationship between (1) the changes in fiber percentages as a result of training and detraining and the percentage of reduction in torque at 30 seconds of recovery and (2) the percentage of reduction in torque at 30 seconds of recovery and maximal aerobic capacity ([Vo.sub.2]max) for the training and detraining subjects and for the 11 additional subjects.

Results

Subjects who underwent endurance exercise training improved their [Vo.sub.2]max, expressed in milliliters per kilogram of body weight per minute, an average of 24% compared with before training (Tab. 2). Subjects who underwent detraining decreased their [Vo.sub.2]max an average of 17% compared with initial values (Tab. 2), with those subjects with the highest initial [Vo.sub.2]max having the greatest decline.[9]

No significant changes occurred in the percentage of type I or total type II muscle fibers as a result of endurance training or detraining (Tab. 2). There were significant changes, however, in the distributions of type IIa and type IIb fibers within the fast-twitch fiber (type II) population as a result of endurance training and detraining (P<.001, Tab. 2). With endurance training, there was a 24% increase in the percentage of type IIa fibers and a 63% reduction in the percentage of type IIb fibers. With detraining, there was a 26% increase in the percentage of type IIb fibers and a 40% decrease in the percentage of type IIa fibers compared with the trained state (Tabs. 1, 2).

The percentage of decline in peak torque after the 1-minute bout of exercise was not altered as a result of training or detraining. The percentage of initial torque after 30 and 60 seconds of recovery, however, was greater after endurance training (Fig. 1a) and lower at 30 seconds of recovery after detraining (Fig. 1b). There were no significant differences in recovery during the second and third minutes of recovery as a result of training or detraining. A positive correlation (r = .75, P<.001) was evident between the percentage of reduction in torque at 30 seconds of recovery and the percentage of type IIb fibers (Fig. 2). A high negative correlation (r = -.84) was evident between the percentage of reduction in torque at 30 seconds of recovery and [Vo.sub.2]max for the training and detraining subjects and for the 11 additional subjects of similar age (Fig. 3).

Discussion

The major finding of this study is that the recovery of peak torque during the first 60 seconds following a standard bout of fatiguing exercise of the quadriceps femoris muscle is inversely related to the percentage of IIb muscle fibers. The vastus lateralis and gastrocnemius muscles both demonstrate the histochemical adaptive changes in type II muscle fibers resulting from training and detraining. The finding of changes only in the histochemical profile in the subpopulations of type II muscle fibers is consistent with that of Andersen and Henriksson,[8] who reported similar increases in type IIa and decreases in type IIb muscle fibers in response to 8 weeks of endurance exercise training.

Previous reports[4,8,14] have suggested that the rate of force recovery is a function of the proportion of type I muscle fibers. The results of our study show that the proportion of type IIb fibers also plays a role, with an increase in the percentage of type lib fibers accompanying a slower recovery of peak torque and a decrease in type IIb fibers being associated with more rapid recovery. Furthermore, our results show a positive correlation between [Vo.sub.2]max and recovery of force following fatiguing isokinetic exercise. The decrease in type IIb fibers and the increase in [Vo.sub.2]max are both components of the adaptive increase in the capacity of skeletal muscle for aerobic metabolism that is induced by endurance exercise training.[15] In this context, the more rapid recovery of torque during the first 30 or 60 seconds after exercise in the trained state could be due to a more rapid regeneration of high-energy phosphates (adenosine triphosphate and phosphocreatine).

Some previous reports have also suggested that force recovery may be greater in muscles with high capillary densities,[16] as well as greater mitochondrial enzyme contents.[14] Capillary density and enzyme levels were not studied on all subjects as part of this study; however, oxidative enzymes decreased, glycolytic enzymes increased, and capillary density remained unchanged after these subjects detrained.[17] Due to the small numbers of subjects, it was not possible to draw conclusions regarding whether individuals with greater proportions of slow-twitch fibers recovered maximal torque output faster than individuals with larger proportions of fast-twitch fibers. Future studies are necessary to determine whether changes in oxidative and/or glycolytic enzymatic activities, capillary density, or high proportions of type I fibers in the quadriceps femoris muscle underlie the differences in the recovery of peak torque after a fatiguing bout of exercise.

We could find no differences in the percentage of decline in peak torque at the end of the isokinetic fatigue test as a result of 12 weeks of endurance exercise training or detraining. Before and after the training or detraining, the percentage of decline in peak torque (ie, the mean peak torque of the 46th-50th knee extensions) ranged from 39% to 60%. This finding suggests the percentage of decline in peak torque at the end of the fatigue bout is not sensitive for describing adaptive changes in the oxidative-glycolytic capacities of the muscle, that is, when the muscle becomes more or less fatigue resistant. The recovery of muscle peak torque was a more sensitive measure, because it improved as a result of endurance exercise training and diminished with detraining. The improved recovery after training seemed to be evident at every 30-second interval for the 3 minutes of recovery. These changes, however, were significantly different only within the first minute of recovery. The first 30 seconds of recovery reflected the relative shifts in oxidative-glycolytic potentials of the fast-twitch pool of muscle fibers (Fig. 2). Similarly, the first 30-second interval of recovery of the quadriceps femoris muscle was inversely correlated to the physiological measure of [Vo.sub.2]max, suggesting a close association with maximal aerobic capacity (Fig. 3).

We believe the results of this study have clinical relevance for the rehabilitation professional. After endurance exercise training types of programs, clinicians may use recovery of peak torque to assess changes in the endurance capacity of the quadriceps femoris muscle. Unlike the percentage of decline in peak torque at the end of a fatigue test (ie, the "fatigue index"), the utility of recovery measures is that they appear to be reflective of and responsive to alterations in physiological factors known to change with endurance exercise training and detraining. Whether recovery indexes reflect alterations in local muscle endurance capacity due to disease or disuse, however, remains to be determined.

Conclusions

These results demonstrate that changes in type IIa and type IIb fiber distributions parallel changes in the rate of torque recovery after a 1-minute fatigue test following 12 weeks of endurance training or detraining. Furthermore, the rate of force recovery after a standard bout of fatiguing exercise is related to maximal aerobic capacity ([Vo.sub.2]max) and may reflect local muscle endurance-exercise adaptations. Using the rate of force recovery after a standard bout of fatiguing exercise, physical therapists can better document the beneficial adaptive responses of skeletal muscle as a result of endurance exercise training or detraining programs.

Acknowledgments

We gratefully acknowledge the support and contributions of Steven J Rose, PhD, PT, FAPTA,(#) who was Associate Professor and Director, Program in Physical Therapy, Washington University School of Medicine, at the time a portion of this study was performed. We also thank Michael J Mueller, PhD, PT, for his help and constructive criticisms. (*) Miles Laboratories Inc, Ames Div, Elkart, IN 46515. (dagger) International Equipment Co, 300 Second Ave, Needham Heights, MA 02194. (double dagger) Cybex, Div of Lumex Inc, 2100 Smithtown Ave, Ronkonkoma, NY 11779. (sections) Perkin-Elmer Corp, 2771 Garey Ave, Pomona, CA 91767/ [parallel] Warren E Collins Inc, 220 Wood Rd, Braintree, MA 02184.

References

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