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korjanc,Faculty of Biology, University of Konstanz, D-78457 Konstanz, Germany
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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To investigate effects of sustained activity on major phenotypic properties, the left extensor digitorum longus muscle of young (15 wk) and aging (101 wk) male Brown Norway rats was subjected to 50 days of chronic low-frequency stimulation (CLFS; 10 Hz, 10 h/day). The contralateral muscle served as control. Changes in metabolic enzymes were analyzed by using glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase as reference enzymes of glycolysis and by using citrate synthase and 3-hydroxyacyl-CoA dehydrogenase as mitochondrial enzymes representative of aerobic-oxidative metabolism. Myosin heavy chain (MHC) isoforms were analyzed by SDS-PAGE. No differences existed between the enzyme activity profiles of control muscles from young and aging rats. CLFS induced similar increases in mitochondrial enzymes, as well as similar decreases in glycolytic enzymes. Although the MHC composition of the control muscles in the aging rats displayed a shift toward slower isoforms, the ultimate changes induced by CLFS led to nearly identical MHC phenotypes in both young and aging rats. These results demonstrate an unaltered adaptability of skeletal muscle to increased neuromuscular activity in the aging rat.
adaptive changes; age; glycolytic enzymes; mitochondrial enzymes; myosin heavy chain isoforms
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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A LARGE BODY OF LITERATURE on age-related changes in skeletal muscle has accumulated during the past years (for reviews see Refs. 13, 20). Major changes comprise a loss of muscle mass due to fiber atrophy and fiber loss, a shift toward slower fiber types, and metabolic changes as mirrored by some reductions in mitochondrial enzyme activities of terminal substrate oxidation. The question has been raised to what extent these alterations are primarily age related or are secondary to the effects of reduced physical activity with aging. In the latter case, enhanced muscular activity should be able to counteract at least some of the above-mentioned changes. Indeed, beneficial effects of various exercise-training protocols have been reported for senescent small mammals and the aging human (e.g., Refs. 3, 15, 19, 24, 26). However, training does not only encompass increased neuromuscular activity but also involves additional effects, e.g., by stress and hormonal factors. A shortcoming of training studies, especially in animals, is the difficulty of performing muscle work under standardized and reproducible conditions. Moreover, high-intensity training is difficult to maintain in animal experiments during prolonged time periods.
Chronic low-frequency stimulation (CLFS) represents an experimental protocol for studying effects of sustained contractile activity under standardized and reproducible conditions. Because it was the aim of the present study to compare the adaptive responses of skeletal muscles in young and aging rats to sustained activity, low-frequency stimulation appeared to be an appropriate experimental protocol. Also, CLFS largely excludes variable parameters such as motivation, habituation, or secondary hormonal effects. Thus it provides a well-defined experimental basis for investigating whether age affects the range of the adaptive responses of skeletal muscle to enhanced contractile activity. An answer to this question would also be of interest for medical applications, e.g., using transformed muscle flaps for cardiomyoplasty (5) or neosphincter construction (1). If aging muscle exhibited restricted adaptive ranges, such applications might be successfully used only in young but not in aging patients.
Numerous studies have shown that CLFS transforms fast-twitch muscles
into slower contracting muscles (for review see Ref. 18). In the adult
young rat, this process encompasses fiber type transitions in the order
of type IIB 
type IID/X type IIA with corresponding
changes in myosin heavy chain (MHC) isoform expression
(11). In addition, CLFS elicits alterations in the metabolic profile of the muscle fibers with decreases in glycogenolytic and glycolytic enzyme activities and increases in mitochondrial enzyme
activities of terminal substrate oxidation (18). These changes improve
the aerobic-oxidative capacity of energy metabolism and lead to an
enhanced fatigue resistance.
The purpose of the present study was to investigate CLFS-induced changes in the myosin isoform pattern and the enzyme activity profile of energy metabolism in extensor digitorum longus (EDL) muscles of young and aging rats and to compare their adaptive ranges under identical conditions of enhanced neuromuscular activity. To this end, EDL muscles of the left hindlimb of 15-wk-old (young adult) and 101-wk-old rats of the Brown Norway (BN) strain were exposed to CLFS for 50 days. After termination of CLFS, their age amounted to 22 and 108 wk, respectively. The average life span (50% survival age) of the BN rats used is ~120 wk (4), and their maximal life span is 158 wk (22). The 108-wk-old animals can thus be considered as late middle aged or old. Their study appeared to be relevant with regard to possible age-related reductions in the adaptive range. We purposely did not include older or senescent BN rats because of the limited survival of such animals.
After cessation of CLFS, stimulated and control muscles from the contralateral leg were analyzed for changes in their MHC isoform composition and their metabolic properties as judged by the activity levels of selected reference enzymes. On the basis of our previous work (2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) served as representative enzymes of the glycolytic pathway, and citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HADH) served as reference enzymes of the citric acid cycle and fatty acid oxidation, respectively.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Animals, stimulation, and muscles. The experiments were performed on young and aging male rats of the BN strain (supplied by the TNO Prevention and Health Center for Ageing Research, Leiden, The Netherlands). The age of the young rats was 15 wk (initial weight 253.3 ± 35.4 g) and that of the aging rats was 101 wk (initial weight 458.0 ± 17.5 g) when CLFS of the left EDL muscle began. CLFS was performed via electrodes implanted laterally to the peroneal nerve of the left hindlimb with a frequency of 10 Hz (21). Stimulation lasted 10 h/day for 50 days. Two experimental groups were studied, 10 young rats and 11 old rats. After 50 days of CLFS, the mean body weight of the young animals (22 wk old) amounted to 281 ± 21.6 g, and that of the old rats (108 wk old) was 374.8 ± 20.5 g. Thus the old group exhibited some weight loss, probably due to reduced food intake during CLFS. However, the animals displayed no signs of discomfort or illness. After cessation of CLFS, the animals were killed, and the EDL muscles from both the stimulated (left) and contralateral (right) hindlimb were excised, weighed, and immersed in liquid N2. One-half of the muscles was used for enzyme activity measurements, and the other one-half was used for electrophoretic analysis of MHC isoform composition.
Enzyme activity determination. Frozen muscle tissue was pulverized under liquid N2 in a small steel mortar and homogenized (1:20, wt/vol) in a cold 100 mM KH2PO4-Na2HPO4 buffer (pH 7.2) containing 2 mM EDTA. The homogenate was sonicated five times for 30 s under intense cooling, stirred for 30 min on ice, and centrifuged for 15 min in a refrigerated Eppendorf centrifuge. The supernatant fraction was decanted, and the pellet was reextracted with the same volume of fresh phosphate buffer used in the first extraction. The two supernatants were combined and used for spectrophotometric activity determination of GAPDH (EC 1.2.1.12), LDH (EC 1.1.1.27), CS (EC 4.1.3.7), and HADH (EC 1.1.1.35), as previously described (2). Enzyme activities were determined at 30°C and expressed as units per gram of fresh weight.
MHC protein electrophoresis. MHC protein isoforms were analyzed by gradient gel electrophoresis as previously described (25). The silver-stained gels were evaluated by integrating densitometry. At least two measurements were performed on each sample.
Statistical analyses. All results are given as means ± SD. A Student's t-test was used to determine whether differences existed between contralateral and stimulated muscles in young rats and aging rats, as well as between contralateral muscles from young and aging rats or stimulated muscles from young and aging rats. The level of significance was set at P < 0.05.
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RESULTS |
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Abstract
Introduction
Methods
Results
Discussion
References
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CLFS-induced changes in mitochondrial enzyme activities. The activity levels of CS and HADH were almost identical in the unstimulated muscles from young and aging rats (Figs. 1 and 2, respectively). CLFS for 50 days led to pronounced increases in both enzyme activities. In the young group, CS increased ~2.6-fold (Fig. 1) and HADH increased ~2.5-fold (Fig. 2). In the old group, CS was elevated ~2.6-fold and HADH ~2.3-fold. After 50 days of CLFS, CS and HADH activities were the same in both young and aging rats.
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CLFS-induced changes in glycolytic enzyme activities. The two reference enzymes of the glycolytic pathway were markedly reduced in the stimulated muscles. In the young group, GAPDH activity decreased to ~62% (Fig. 3) and LDH to ~55% (Fig. 4) of the corresponding control values. In the old group, the stimulation-induced decreases were similar, with GAPDH and LDH having decreased to ~56% (Fig. 3) and ~52% (Fig. 4) of the respective controls. Thus GAPDH and LDH reached similar activity levels in young and old muscles after 50 days of CLFS.
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MHC isoform patterns of normal EDL muscles in young and aging rats. According to their MHC isoform composition, unstimulated EDL muscles displayed a typical fast MHC pattern. The slow MHC I isoform amounted to only ~6% in the young and ~5% in the aging rats (Fig. 5). MHC IIb, MHC IId, and MHC IIa predominated, but their distribution differed between control muscles from young and aging rats. MHC IIb was the major isoform (~38%) in the young group, whereas MHC IId was the most prominent isoform (~47%) in the old group. MHC IIa was slightly higher in the old group than in the young group, but this difference was not significant. Collectively, these differences reflected an age-related fast-to-slow shift within the pattern of the fast MHC isoforms.
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CLFS-induced changes in MHC isoform distribution. Fifty days of low-frequency stimulation produced fast-to-slow transitions that, however, were restricted to the fast MHC isoforms in muscles from both young and aging rats (Figs. 6 and 7). MHC IIa was the predominant isoform in 50-day-stimulated muscles from young and aging rats. In the young group, the relative increase in MHC IIa was paralleled by similar decrease in MHC IIb and to a lesser extent by a decrease in MHC IId. In the old group, the increase in MHC IIa corresponded to pronounced decreases in both MHC IIb and MHC IId. Minor increases in MHC I were observed in stimulated muscles from both young and aging rats, but even after 50 days the relative concentrations of MHC I amounted to only ~12 and ~8%, respectively.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Although several studies suggest that exercise training evokes similar changes in muscles from young and middle-aged/old animals or humans (3, 6, 7, 10, 12, 16, 24), the question as to age-related changes in the adaptive range of skeletal muscle remains open. The reason is that exercise-training studies using humans lack the possibility of comparing genetically identical groups of young and old individuals. This possibility is provided by animal studies in which different age groups from genetically defined strains can be exposed to identical training protocols. However, studies on animals have the disadvantage that it is difficult to perform high-intensity training or even to attain maximal performance during long time periods (3, 8). Because of their heavier body weight and other factors, this applies especially to aging animals being less motivated and less capable than younger animals (9). These considerations have prompted us to use CLFS as a standardized regimen of maximally increased contrac- tile activity to compare adaptive changes in young and aging rats of a genetically defined inbred strain.
The results of our enzyme studies confirm previous observations that aging does not result in a decline in the aerobic-oxidative capacity of muscle energy metabolism (27, 28) and that exercise training evokes similar increases in mitochondrial enzyme activities of muscles from young and aging rats (28). Thus HADH and CS activities increase to same or similar levels in the stimulated EDL muscles of young and aging rats. Identical adaptive ranges also apply to the investigated reference enzymes of the glycolytic pathway. Under the influence of CLFS, GAPDH and LDH activities decrease from similar starting points to similar levels in young and aging rats.
Our results disagree to some extent with previously published findings on the effects of CLFS on a fast-twitch muscle in aging rats (27). In that study, the flexor digitorum longus muscle of 24- to 32-wk-old and 104- to 112-wk-old F344 rats was subjected to CLFS (8 h/day) for up to 90 days. The increase in CS activity in the old animals was found to be smaller than in the young animals, reaching only 60% of the latter after 50 days of CLFS. Except for the use of a different muscle and a shorter duration of the daily stimulation protocol, we have no explanation for the discrepancy between that study (27) and our results. The possibility that a smaller increase in CS activity resulted from the use of rats from a different strain is less likely because our present findings agree with results from a previous study in which we investigated the effects of CLFS in adult male Wistar rats (21). Thus CS activity increased 2.6-fold in the 50-day stimulated EDL muscle (BN rats), whereas it increased 2.5-fold in the 35-day-stimulated tibialis anterior muscle (Wistar rats).
The present findings confirm previous observations on age-related differences in myosin composition of muscles from young and old rats, i.e., an age-related shift from MHC IIb toward MHC IIa (14, 23, 24). Therefore, the CLFS-induced changes originate from different starting points of the MHC isoform pattern, i.e., from a predominance of MHC IIb in the muscles from young rats and from a predominance of MHC IId in the muscles from older rats. It is remarkable that, independent of these differences, CLFS leads to similar MHC isoform patterns in the muscles from the 22-wk-old and 108-wk-old rats. Thus, with the exception of a slightly higher increase of MHC I in the young group, the relative concentrations of MHC IIb, MHC IId, and MHC IIa are very similar in the stimulated muscles from young and aging rats (Fig. 8). The finding that 50 days of CLFS led to only small increases in MHC I is in agreement with results from previous studies on chronically stimulated rat fast-twitch muscles (11, 17, 25) and emphasizes the notion that, contrary to larger animals, rat fast-twitch muscle displays some sort of resistance to a full fast-to-slow conversion.
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In summary, we show that sustained contractile activity as induced by increased neuromuscular activity in fast-twitch muscles from 22-wk-old and 108-wk-old BN rats leads to similar changes in enzyme activities of anaerobic and aerobic energy metabolism, as well as to similar transitions in the MHC isoform profile. Our findings, therefore, support the notion of unaltered adaptive ranges during aging.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ted Putman for discussion and Elmi Leisner for excellent technical assistance in the animal stimulation.
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FOOTNOTES |
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This study was supported by the Deutsche Forschungsgemeinschaft (Pe 62/25-1).
Address for reprint requests: D. Pette, Faculty of Biology, Univ. of Konstanz, D-78457 Konstanz, Germany (E-mail: dirk.pette{at}uni-konstanz.de).
Received 8 December 1997; accepted in final form 21 April 1998.
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