INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

AGING IS COMMONLY ASSOCIATED with a decrement in motor function due, in part, to a loss in muscular strength and fatigue resistance. In turn, these decrements are reflected in detrimental adaptations in some of the mechanical, biochemical, and morphological properties of the skeletal musculature (9, 12, 13, 20). Some of these adaptations appear to be related to a reduction in use, i.e., decreased levels of neuromuscular activity (26), as is typical of muscles in the lower extremities of old people and animals (9). These adaptations, however, also have been associated with intrinsic age-related changes in the muscles themselves (12).

Skeletal muscles in old individuals are responsive to increased levels of neuromuscular activity. For example, progressive resistance training results in strength gains due to either muscle hypertrophy (5, 25) and/or neural factors (24) in older people. The capacity for muscle hypertrophy is attenuated in older subjects (33). The reasons for the attenuated cellular and biochemical responses of atrophied muscles of older individuals to resistance exercise remain unclear. Heavy resistance exercise can induce inflammation and/or damage in the muscle, and the associated degeneration-regeneration responses can be beneficial for a subsequent increase in muscle mass (19). In addition, there appears to be an age-related impairment in myogenic potential as indicated by a deficiency in the capacity for skeletal muscle reinnervation in old rats (8, 23).

Heavy resistance exercise, such as weight lifting, is easy to apply in humans but is very difficult to apply in laboratory animals. Human studies on the cellular responses to resistance training are limited due to the invasive nature of muscle biopsies and to the risk of using old people as subjects, as well as the more limited control of lifestyles. To circumvent these problems, we have been using a weight-lifting protocol designed for rats (30). Recently, we have reported morphological and biochemical evidence of muscle fiber hyperplasia associated with muscle damage and regeneration in the plantaris (Plt) muscle after a single exhaustive session of weight lifting in previously nontrained young rats (28).

The purpose of the present study was to compare the myogenic response of hindlimb muscles in young (14-20 wk of age) and old (>120 wk of age) rats with a single intense session of weight-lifting exercise. We hypothesized that the myogenic potential of old rats after a single bout of exercise is less than that in young rats. Serum creatine kinase (CK) activity was used as a measure of muscle damage. Changes in the incorporation of [³H]thymidine and [¹⁴C]leucine in selected hindlimb extensor and flexor muscles after the weight-lifting session were followed for 2 wk to determine the level of mitotic activity and amino acid uptake, respectively. The expression of myogenic determination genes (MyoD; protein and mRNA levels) and 5-bromo-2'-deoxyuridine (BrDU) labeling also were used as indicators of cell proliferation and cellular mitotic activity.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. The following two groups of Wistar male rats (specific pathologen free) were studied: 1) young (14-20 wk old; 380-520 g body mass; total n = 46) rats and 2) old (>120 wk old; 375-540 g body mass; total n = 37) rats. Exercised (see below) and nonexercised subgroups were studied in both age groups. In addition, 3-wk-old rats (85-95 g body mass; total n = 12) were used as positive controls for the analysis of muscle-specific gene expression (see below). The animals were housed in standard cages and were provided food and water ad libitum. The room temperature was kept at 23 ± 1°C, and a 12:12-h light-dark cycle was maintained throughout the experiment. All experimental procedures were conducted in accordance with the Japanese Physiological Society Guide for the Care and Use of Laboratory Animals as approved by the Tokai University School of Medicine Committee on Animal Care and Use and followed the American Physiological Society Animal Care Guidelines.

Exercise protocol. The hindlimbs of the rats in the exercise groups were trained for one intense exercise session using a weight-lifting technique described in detail elsewhere (28, 30). The exercise was performed preferentially by the plantarflexors, with a minimal usage of the dorsiflexors. The session involved multiple sets of 10 repetitions (lifts) per set with ~1 min rest between each set. The first set of lifts was with a 500-g load. In the subsequent sets, an additional 500-g load was added until the rat could not complete 10 repetitions. The load then was adjusted in 100-g increments and/or decrements until the maximum load at which 10 repetitions could be completed, i.e., the 10 repetition maximum (10 RM), was determined. The 10 RM was repeated until the rat could not complete the set, and then the load was decreased by 500 g. This procedure was followed until the rat failed to complete three consecutive sets, even when the load was being reduced. The total time of the exercise bout was ~30-40 min for the young and ~20-30 min for the old rats. The 10 RM (g), number of sets, and total amount of load lifted (number of lifts × load lifted by different groups in absolute values expressed as kg) were recorded.

Measurement of serum CK activity. Blood samples (0.2 ml) were obtained from the caudal vein before and 30 and 60 min after the exercise session. Serum CK activity was measured using a standard kit (Monotest CK-NAC; Boehringer, Mannheim, Germany) and was used to estimate exercise-induced muscle damage. The activities were expressed as international units per milliliter.

Muscle fiber number. To determine whether a decrease in fiber number occurred during aging, the number of total and branched fibers of the Plt muscles in both groups were counted using a nitric acid digestion method (29, 30). Briefly, the frozen muscle was thawed in distilled water for a few minutes and then immersed in 15% nitric acid (Wako Pure Chemical, Osaka, Japan) for 2-3 h at room temperature. The muscle was washed in cooled distilled water for 60 min at 4°C. The solution was changed from distilled water to 0.01 M PBS (pH = 7.4) and was stored at 4°C. Subsequently, a few muscle fiber bundles were transferred to a silicone-coated petri dish containing 0.01 M PBS (pH = 7.4) at room temperature. All fibers were teased free, and the total number of straight (nonbranched) and branched fibers was carefully and precisely counted under a dissection microscope (×10-15). These procedures were repeated until all fibers, nonbranched and branched, in each muscle were counted.

Histochemical and immunohistochemical analyses. Histochemical and immunohistochemical analyses were performed 48 h after the exercise session in exercised (n = 4) and nonexercised (n = 2) rats in both groups. BrDU (Takeda Chemical, Osaka, Japan), a nonradioactive marker for DNA synthesis, was injected (100 mg/kg ip) 1 h before sampling. This procedure is useful for labeling proliferating cells in muscle (28, 29). The rats were killed with an overdose of pentobarbital sodium (60 mg/kg ip), and the soleus (Sol) and Plt muscles of both hindlimbs were excised within 10 min. The Sol and Plt muscles then were wet weighed after trimming excess connective tissue, were immediately frozen in isopentane precooled by liquid nitrogen, and were stored at 80°C. After equilibration at 20°C, each muscle was divided longitudinally into three (Sol) or six (Plt) portions to allow full characterization of the entire muscle (29). Serial 10-µm-thick transverse sections (10-20 sections) were cut from each muscle portion. Staining of hematoxylin-eosin and actomyosin ATPase (mATPase) after acid (pH 4.3) preincubation was performed on four to eight sections to examine the general morphology and the fiber type characteristics of the muscles.

Western blotting procedures. To determine the level of MyoD expression, the Sol and Plt muscles from two nonexercised rats from the young and old groups were prepared for Western blotting. In addition, muscles from two 3-wk-old rats were analyzed as a positive control. Each muscle was minced using scissors at 4°C on an ice-cooled glass plate. The samples were immersed in a homogenizing buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS in PBS, and 0.1 mM phenylmethylsulfonyl fluoride was added at the time of use at 4°C. Muscle tissues were homogenized (×10) with a glass homogenizer and were incubated for 30 min at 4°C. Each homogenate was transferred to microfuge tubes and centrifuged at 15,000 g for 20 min at 4°C, and the supernatant was obtained. Cell lysate with an equal volume of electrophoresis sample buffer (0.15 M Tris · HCl, 4% SDS, 10 mM EDTA, 40% glycerol, and 10% 2-mercaptoethanol, pH 6.7) was boiled for 5 min, separated by 10% SDS-PAGE, and blotted for 1.5 h with a constant current of 150 mA on a polyvinylidene difluoride membrane (ATTO, Tokyo, Japan). In this step, the total protein concentration of each sample was adjusted preliminarily. The membrane was blocked with a blocking buffer (5% nonfat milk powder in 0.01 M PBS, pH 7.4, containing 0.1% Tween 20) overnight at 4°C. Primary antibody [polyclonal anti-MyoD (C-20) from rabbit; Santa Cruz Biotechnology, Santa Cruz, CA] was applied in a dilution of 1:100 with blocking buffer for 1.5 h at 37°C. The blot was washed for 1.5 h with several changes of PBS containing 0.1% Tween 20 (PBS-Tween) at room temperature and then was incubated with a second antibody [anti-rabbit IgG F(ab')₂ combined with sheep horseradish peroxidase; Amersham International] diluted 1:5,000 with blocking buffer for 1.5 h at 37°C. The blot was washed with PBS-Tween for 2 h. Antibody binding was visualized by the enhanced chemiluminescence (ECL) technique (ECL+plus System; Amersham International) and X-ray film (BioMax MS-1; Kodak, Tokyo, Japan). The immunoblots were photographed and scanned with a scanning densitometer (Ultrascan laser; Pharmacia LKB, Uppsala, Sweden) for quantification.

RT-PCR analyses. Total RNA was extracted from nonexercised young (n = 2), old (n = 2), and 3-wk-old (n = 10) rats using the guanidine isothiocyanate technique (10). The Sol and Plt muscles were dissected from each rat, the tendons were removed, and the muscles were minced using scissors at 4°C on an ice-cooled glass plate under the RNase-free condition. Each muscle was lysed in guanidine isothiocyanate buffer and homogenized using a polytron (Kinematica Littau/Luzen, Switzerland). The lysate was layered on a CsCl gradient buffer (5.7 M CsCl/3 M sodium acetate, pH 6, sterile), spun in an ultracentrifuge at 179,000 g (SW55; Beckman, Fullerton, CA) for 21 h at 20°C, and then prepared for extraction of intact total RNA. RNA quantity was determined by an optical density measurement at 260 nm. Samples were stored at 80°C. Primer pairs and the nucleotide position used for PCR amplification of MyoD mRNA were as follows: MyoD-forward, 5'-ACCATGGAGCTACTATCGCCGCCA-3', nucleotide 2590-2611 (32); MyoD-reverse, 5'-TCTCGAGCACCTGGTAAATCGGATT-3', complementary to nucleotide 4287-4307 (32). RT-PCR was performed using an RNA-PCR kit (Perkin-Elmer, Cetus, CT). To enable a comparison between muscles from young, old, and 3-wk-old rats, 25, 30, and 35 PCR cycles were chosen.

Analyses of mitotic activity and amino acid uptake. Our previous data indicate that in vivo [³H]thymidine and [¹⁴C]leucine labeling is a useful method to detect the mitotic activity and amino acid uptake in the muscles after the exercise (28). Analyses of mitotic activity and muscle amino acid uptake were performed at rest and 1-4, 7, 10, and 14 days after exercise in young (n = 31, 3-6/time point) and old (n = 24, 3/time point) rats. [³H]thymidine (methyl-³H; 15.5 MBq/kg ip; specific activity 247.9 GBq/mmol; NEN Life Science Products, Boston, MA) and [¹⁴C]leucine (U-¹⁴C; 1.15 MBq/kg ip; specific activity 13.6 GBq/mmol; NEN Life Science Products) were injected 1 and 3 h before sampling to label proliferating cells and proteins that use leucine during protein synthesis, respectively. The rats were overdosed with pentobarbital sodium (60 mg/kg ip), and the following muscles were removed bilaterally: primary extensors [Sol, Plt, and gastrocnemius (Gas)] and primary flexors [tibialis anterior (TA) and extensor digitorum longus (EDL)]. After excess connective tissue and fat were removed, each muscle was wet weighed and homogenized in 0.02 M phosphate buffer (pH = 7.4) at a 1:20 dilution at 4°C. In the next step, 1 ml of the homogenate from each muscle sample was added to 5 ml of 10% TCA and mixed well. This mixture was centrifuged (2,050 g for 10 min), and the upper solution (TCA) was removed. This procedure was repeated five times, and the remaining TCA-insoluble material was collected and dried with 70% ethanol. The dried material was treated overnight with 1 ml of dissolving solution (Solvable; Packard, Meriden, CT) at 45°C, and then a 10-ml liquid scintillation cocktail (Atomlight; Packard) was added to count radioactivity (Beckman LS4800). The total protein concentration in each homogenate was measured, and the radioactivity of each sample was expressed as disintegrations per minute per milligram protein.

Analysis of myosin synthesis. The determination of myosin synthesis was performed at the same time points and for the same groups as for the analysis of mitotic activity and amino acid uptake. Myosin was extracted with 0.6 M KCl solution (50 ml) from a 1-ml homogenate for 15 min at 4°C and was filtered with three sheets of gauze. The myosin-extracted KCl solution was diluted with cool distilled water (1:20), which resulted in the reappearance of myosin deposits. The diluted solution was passed through an omnipore nondissolving membrane filter (10 µm aperture and 47 mm diameter; Nihon Millipore, Yonezawa, Japan). The membrane containing the deposits was dried, cut into several pieces, and soaked in a dissolving solution overnight at 45°C. The radioactivity was counted using the same procedures employed for the mitotic activity and protein synthesis analyses. Values were expressed as disintegrations per minute per milligram protein.

Statistical analyses. All data are expressed as means ± SE. Differences in body mass, muscle mass, fiber numbers, and [³H]thymidine and [¹⁴C]leucine uptake levels between young and old rats were determined using Student's t-tests. ANOVA was used to determine overall differences, and Duncan's post hoc analyses were used for individual group differences for the preexercise and serial postexercise data for [³H]thymidine, [¹⁴C]leucine, and myosin fraction uptake. Standard regression analysis and Pearson's product correlation procedures were used to determine the relationship between [³H]thymidine uptake and CK activity. Differences were considered statistically significant at either the 0.05 or the 0.01 levels.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Exercise capacity. The total load lifted during the single exhaustive exercise bout was lower in old than young rats. The total number of sets, the 10 RM, and the total amount of load lifted were 24 ± 3 and 13 ± 5 sets, 2,664 ± 38 and 2,083 ± 42 g, and 417 ± 86 and 209 ± 26 kg for the young and old rats, respectively. All values were significantly higher in the young than the old rats (P < 0.01).

Body mass, muscle mass, and total muscle fiber number. The mean final body mass was not significantly different between the two groups (Table 1). However, it should be noted that the body masses of the old rats had increased to 650-750 g and then gradually decreased with advancing age. The mean absolute masses of the TA, Plt, and Gas and relative masses of TA, Sol, Plt, and Gas were significantly smaller in old than young rats. The mean total fiber number in the Plt muscle was significantly lower (17%) in old than young rats (Table 2). Although the absolute number of branched fibers was 24% (P > 0.05) higher in the old than the young rats, the incidence of branched fibers relative to nonbranched fibers was low in both young (0.26%) and old (0.38%) rats.

Serum CK activity. The mean preexercise serum CK levels were similar in young (n = 25) and old (n = 21) rats (Fig. 1). These levels were significantly increased in both groups 30 or 60 min after the exercise session. The postexercise values were significantly lower in the old than the young rats at both time points.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Mean serum creatine kinase (CK) activities at rest and 30 and 60 min after a single exhaustive resistive bout of exercise. Bars are SE. IU, international units. Rest, preexercise. * P < 0.05 (young vs. old rats);  P < 0.05 and P < 0.01 (rest vs. postexercise).

Muscle morphology and immunostaining. In contrast to the typical mosaic pattern of fiber types (slow fibers dark, based on mATPase staining at a pH 4.3 preincubation) in the Plt muscle of young rats (Fig. 2A), "fiber type grouping," i.e., an abnormal number of fibers of the same ATPase type that are adjacent, was observed in the Plt of old rats (Fig. 2B). In addition, fibers having unusual cross-sectional shapes, e.g., small angulated fibers (Fig. 2C), were evident in all muscles of old, but not young, rats.

View larger version (99K): [in this window] [in a new window]   Fig. 2.   Representative cross-sectional profiles of the plantaris muscles of a young (A) and an old (B and C) rat. A and B: actomyosin ATPase at a preincubation of pH 4.3. C: hematoxylin and eosin (HE) staining. Fiber type grouping (B) and small angulated fibers (arrowheads in C) were evident in muscles of old, but not young (A), rats. The appearance of small angulated fibers is consistent with degenerating fibers that are not reinnervated (3, 21, 22). Fiber type grouping of slow fibers in older animals (14) has been interpreted as evidence for the reinnervation of some of the fast fibers by motoneurons innervating slow muscle fibers (12, 16). Calibration bar is equal to 100 µm. Magnifications: A and B, ×24; C, ×120.

View larger version (124K): [in this window] [in a new window]   Fig. 3.   Representative cross-sectional profiles of the plantaris (A-F) and soleus (G and H) muscles of a young (A, C, E, and G) and old (B, D, F, and H) rat. A and B: HE. C and D: anti-5-bromo-2'-deoxyuridine (BrDU). E-H: anti-myogenic determination gene (MyoD) staining. Arrowheads in A and B indicate necrotic (damaged) fibers, arrowheads in D indicate cells showing positive staining for BrDU (cell proliferation), and arrowheads in F and H indicate positive staining for MyoD (myoblast). Calibration bars are equal to 100 µm. Magnifications: A and B, ×100; C, D, G, and H, ×120; E and F, ×110.

Expression of MyoD protein and mRNA. An immunoreactive band of 35 kDa corresponded to the predicted molecular mass of the rat MyoD protein (Fig. 4). The lowest levels of MyoD protein were observed in both the Sol and Plt of old rats. To examine the age dependence of the MyoD expression, we compared the nonexercised young and old rats with 3-wk-old rats. MyoD protein tended to be higher in the 3-wk-old than in the young rats for the Plt but not the Sol muscles. The expression of MyoD mRNA levels in both the Sol and Plt was highest in the 3-wk-old rats and lowest in the old rats (see 25 and 30 cycles; Fig. 5). The expression levels were lowest in the Sol of old rats (Fig. 4). It also appeared that the expression of MyoD mRNA levels were higher in the Plt than the Sol muscle in all groups. We confirmed that the nucleotide sequence of the RT-PCR product was completely matched to the MyoD mRNA (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Resting levels of MyoD protein contained in the soleus (Sol) and plantaris (Plt) muscles of a representative old, young (Y), and 3-wk-old (3w) rat analyzed by Western immunoblot. An immunoreactive band of ~35 kDa (K) corresponded to the predicted molecular mass of the rat MyoD protein. The density of each band was expressed relative to the background density (considered to be 1.0). These values were 2.97, 9.67, and 8.41 for the Sol and 1.30, 3.43, and 14.18 for the Plt in old, Y, and 3w rats, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   MyoD mRNA in the Sol and Plt muscles of old, young (Y), and 3-wk-old (3w) rats measured by RT-PCR. Kb, kilobase.

Thymidine and amino acid uptake rates. The flexor muscles (TA and EDL) of old rats had a significantly lower uptake of [³H]thymidine compared with young rats in the resting state (Table 3). The extensor muscles (Sol, Plt, and Gas) in young and old rats were similar in thymidine uptake. There was no significant difference between young and old rats in [¹⁴C]leucine uptake in either the extensor or flexor muscles. Significant increases in the uptakes of [³H]thymidine and [¹⁴C]leucine in the extensors of young rats were observed at 2 and 10 days after exercise, respectively, whereas there was no significant change in the extensors of the old rats (compare Figs. 6 and 7). In addition, no significant changes in the uptake of either [³H]thymidine or [¹⁴C]leucine were observed in the flexor muscles of young or old rats after the single bout of exercise (Figs. 6 and 7). Because there was no exercise effect on the overall mean uptakes for the flexor muscles for either group (Figs. 6 and 7), we then normalized the exercise effects of the extensors to the flexor muscles and compared the response of young and old rats (Fig. 8). This procedure minimized the intra-animal variability. The uptake of [³H]thymidine was elevated significantly at 2 days after exercise in young rats (Fig. 8A). In contrast, there was a small insignificant peak at 1 day after exercise in old rats (Fig. 8A). [¹⁴C]leucine uptake in the young rats was elevated up to 10 days after exercise (significant differences at 3 and 10 days) and then returned to rest levels at 14 days. In the old rats, the uptake pattern was similar to that of the young rats for up to 7 days but returned to resting levels 10 days after exercise and remained at that level up to 14 days (Fig. 8B).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Absolute change in [3H]thymidine (A) and [14C]leucine (B) uptake in extensors and flexors during the 2 wk after a single exhaustive bout of weight lifting in young rats. Values are expressed as means ± SE. Extensors, Sol, Plt, and gastrocnemius; flexors, tibialis anterior and extensor digitorum longus. Rest, values from nonexercised control rats. ** Significantly different from rest at P < 0.01.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Absolute change in [3H]thymidine (A) and [14C]leucine (B) uptake in extensors and flexors during the 2 wk after a single exhaustive bout of weight lifting in old rats. Values are expressed as means ± SE. Extensors, Sol, Plt, and gastrocnemius; flexors, tibialis anterior and extensor digitorum longus. Rest, values from nonexercised control rats. Note that there are no significant changes during the 2 wk after the exercise bout.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Changes in [3H]thymidine (A) and [14C]leucine (B) uptake during the 2 wk after a single exhaustive bout of weight lifting. Uptake values were calculated as (mean value of extensors)  (mean value of flexors) in both legs and are expressed as means ± SE. Extensors, Sol, Plt, and gastrocnemius; flexors, tibialis anterior and extensor digitorum longus. Rest, values from nonexercised control rats. * and ** Significantly different from rest at P < 0.05 and P < 0.01, respectively.

Fraction of amino acid uptake for myosin. The uptake of [¹⁴C]leucine for the myosin fraction for the extensor muscles was elevated significantly at 10 days after exercise for the young rats, whereas there were no changes at any other postexercise time point for the young or old rats (Fig. 9A). When the uptake of [¹⁴C]leucine for the myosin fraction on the extensor muscles was expressed relative (%) to total uptake, there was a progressive decrease in old rats that reached significance at 7, 10, and 14 days postexercise (Fig. 9B). The resting levels for the relative uptake of [¹⁴C]leucine for the myosin fraction were similar in the young and old rats, i.e., 29 ± 3 and 26 ± 4%, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 9.   Changes in the absolute [14C]leucine uptake for the contractile component (myosin) of the extensor muscles (A) and its fraction (%) relative to total uptake for the muscle (B) during the 2 wk after a single exhaustive bout of weight lifting. Rest, values from nonexercised control rats. Values are expressed as means ± SE. * and ** Significantly different from rest at P < 0.05 and P < 0.01, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Muscle damage and appearance of proliferating cells and myoblasts. Although the serum CK activities 30-60 min after the exercise bout were increased significantly in both the young and old rats, the increase was two- to threefold higher in the young rats (Fig. 1). With the use of the same weight-lifting model, the degree of CK leakage clearly reflected the severity of damage in exercised muscles of young rats (28). In the present study, in the young rats, these changes were closely associated with the incidence of degenerated fibers in the histological sections of the Plt and Sol muscles (Fig. 3, A and G) and by the increased uptake of [³H]thymidine and [¹⁴C]leucine in the extensor, but not the flexor, muscles postexercise. These changes were not present or were of a lesser magnitude in the old rats. The similarities in the number and location of anti-BrDU and MyoD positive cells in the Plt and Sol muscles 2 days after exercise in young rats suggest that some of the cells that were proliferating were myogenic cells. The number of these cells at the same time point was lower in old than young rats (Fig. 3, C-H), indicating a reduced proliferating capacity of myogenic cells in muscles of old rats. The lower levels of MyoD protein (Western blots) and mRNA (RT-PCR) in the Sol and Plt of old compared with young and 3-wk-old rats are consistent with a decreased reparative capacity in old rat muscles (Figs. 4 and 5).

Thymidine uptake and activity of myogenic cells. There was a significant peak in [³H]thymidine uptake 2 days after the exercise bout in young, but not old, rats (Fig. 8A). Recently, we demonstrated that this time point also shows peak proliferation of satellite cells for the regeneration associated with damaged muscle fibers, to include fiber hyperplasia, after a weight-lifting bout in young rats (28). Furthermore, there was a significant correlation between the serum CK activity 30-60 min after the exercise bout and the uptake of [³H]thymidine 2 days after the exercise bout in young, but not old, rats (Fig. 10). These results also indicate that the ability of the muscle to initiate reparative processes is reduced in old compared with young rats. An impaired capacity for muscle regeneration in old animals also has been reported after bupivacaine injection (8), contraction-induced injury (12), ischemic necrosis (31), eccentric contraction-induced injury (4), and transplantation of muscle grafts (7).

View larger version (14K): [in this window] [in a new window]   Fig. 10.   Relationship between serum CK activity and [3H]thymidine uptake in the extensor muscles in young and old rats after a single exhaustive bout of weight lifting. Abscissa, CK leakage 30-60 min after exercise; ordinate, [3H]thymidine uptake 2 days after exercise. The regression line and correlation for the young rats are shown, P < 0.01. There was no relationship between these two variables in old rats.

Amino acid uptake and ability for muscle hypertrophy. Young rats showed a biphasic response in [¹⁴C]leucine uptake, i.e., peaks at 1-4 and at 10 days after the exercise bout (Fig. 8B). The first peak most likely reflects increased protein synthesis (hypertrophy) in the primary tissues (contractile proteins, connective tissue, revascularization, activation of satellite cells, etc.), whereas the second peak may reflect the synthesis of the contractile components of regenerated and/or de novo muscle fibers (hyperplasia; see Ref. 28). Previous studies have demonstrated that muscle protein synthesis accelerates after the formation of myotubes to allow for accumulation of the contractile components within the cytoplasm and differentiation of the myofibers (15). The old rats showed a similar peak in [¹⁴C]leucine uptake at 1 wk but no peak at 2 wk after the exercise bout (Fig. 8B). Furthermore, the postexercise data for [¹⁴C]leucine uptake for myosin (Fig. 9A) in the extensor muscles and for the fraction of myosin uptake relative (%) to the total uptake (Fig. 9B) suggest that the increase in amino acid uptake during the first week in old rats was used primarily for the synthesis of noncontractile protein such as connective tissue. An age-related decrease of myosin heavy chain synthesis rate has been reported by Balagopal et al. (2), suggesting a decreased capacity for hypertrophy in muscles of old rats. Combined with the small increase in [³H]thymidine uptake in old rats, these results support the view that there is a lower muscle capacity for hypertrophy, including hyperplasia, in old than in young rats.

Perspective