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Fiber Atrophy and Hypertrophy in Skeletal Muscles of Late Middle-Aged Fischer 344 x Brown Norway F1-Hybrid Rats

¹ Faculty of Kinesiology
² Faculty of Medicine, University of Calgary, Alberta, Canada.

	Abstract

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We examined young adult and late middle-aged male rats to test the hypothesis that gastrocnemius (a locomotor muscle) demonstrates reduced fiber size with aging, whereas soleus (a postural muscle) demonstrates atrophy of some fibers and compensatory hypertrophy in other fibers. Although body mass was greater in late middle-aged animals, mass was reduced in gastrocnemius but not soleus muscle. In another group of animals, physical activity was reduced by 34% in late middle-aged animals. Whereas mean fiber size was lower in gastrocnemius of late middle-aged animals, it was not different in soleus. Histograms revealed atrophied fibers (1000 µm²) in soleus and gastrocnemius and hypertrophied fibers (8000 µm²) in soleus with aging. Atrophied fibers often demonstrated no subsarcolemmal mitochondrial staining, suggesting denervation, whereas hypertrophied fibers often demonstrated cytochrome oxidase deficiency, suggesting mitochondrial dysfunction. These results underscore the divergent influences (e.g., physical inactivity, denervation, mitochondrial dysfunction) affecting fiber size with aging.

Note that the measurements in each of these prior studies compared the mean fiber cross-sectional areas between age groups and this approach conceals an increasing heterogeneity of fiber size within individual muscles with aging. For example, Lexell and Taylor (3) noted that the range of fiber cross-sectional area was greater in vastus lateralis muscles from older humans, characterized by an increase in frequency of both atrophied and hypertrophied muscle fibers in the same muscles. Furthermore, in a follow-up study of physically active older participants over an 11-year period, Aniansson and colleagues (4) found an increase in mean fiber cross-sectional area between the ages of 76 and 80 years, which was interpreted as a compensation for a reduced number of motor units in these physically active individuals. These results (4), and those of Lexell and Taylor (3), suggest that both fiber atrophy and hypertrophy may occur in skeletal muscle with aging. Interestingly, a recent study in cardiac tissue has revealed a similar pattern (atrophy and hypertrophy) in senescent hearts of F344BN rats (14), consistent with the idea that this pattern occurs in muscles that remain highly activated with aging. In this respect, the specific nature of the alterations in fiber size likely reveals important clues to the cause, and thus potential treatment, of whole-muscle atrophy with aging. For example, fiber atrophy could occur due to reduced fiber activation secondary to a lower amount and/or reduced intensity of daily locomotor activity with advancing age, as seen in disuse models such as hindlimb suspension (15–17). Denervation, which is associated with aging skeletal muscles (18), would also result in fiber atrophy (19,20). On the other hand, recent evidence shows that focal atrophy along the length of individual myocytes in aged muscles coincides with regions of mitochondrial dysfunction in three-dimensional reconstructions from serial cross-sections (21,22), showing that fiber atrophy may also be a manifestation of mitochondrial DNA damage with aging.

Based on these previous findings, we reasoned that there would be variable alterations in fiber size with aging in soleus muscle (a postural muscle and thus less affected by reduced physical activity with aging), and gastrocnemius muscle (a locomotor muscle expected to exhibit reduced activity with aging), due to intermuscle and intramuscle variation in both the degree of mitochondrial dysfunction or denervation, and the relative extent to which the muscles or muscle regions experience a reduction in activation with aging. Specifically, we hypothesized that the gastrocnemius muscle would demonstrate fiber atrophy due mainly to reduced physical activity with aging, whereas the soleus muscle would demonstrate a fracture in the fiber size distribution due to selective atrophy of fibers afflicted with mitochondrial dysfunction or denervation, and compensatory fiber hypertrophy in other muscle fibers.

	METHODS

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Animals
Six 8-month-old and six 28–30-month-old male F344BN rats were obtained from the National Institute on Aging (NIA) and housed in pairs in cages with filter bonnets at the University of Calgary Medical School vivarium prior to study. Note that one of the 28–30-month-old animals and three of the 8-month-old animals were included in a recently published study of the skeletal muscle aerobic metabolic performance in these animals (12). Although it is acknowledged that there are developmental differences between humans and rats that preclude an exact relative age comparison, the ages of the animals were selected to represent young adult (8 months old) and late middle (28–30 months old) age, based on published survival characteristics for this strain of rat (13) in a relative comparison to survival rates in humans (23). Animals were studied within 3 weeks of arrival from the NIA colony. Necropsies were performed at the end of each experiment to prevent contamination of the data by inclusion of diseased animals, as suggested by NIA guidelines (24). None of the animals in this data set were omitted on this basis.

Surgical Procedures
All procedures were conducted with approval from the University of Calgary Animal Care Committee. Animals were anesthetized with sodium pentobarbital (75 mg/kg i.p.) and prepared for removal of the soleus and gastrocnemius muscle from the right leg. Briefly, this involved removing the skin from the right leg, ligating the femoral artery to prevent excessive bleeding, and carefully dissecting out the gastrocnemius, plantaris, and soleus muscles. Muscles were separated from one another, and the fat and connective tissue were removed prior to weighing. After surgery, animals were euthanized with 25 mg sodium pentobarbital injected into the heart. A cross-section through the midbelly of the soleus muscle [containing mainly slow oxidative fibers (25)] and gastrocnemius muscle [which contains both a highly glycolytic region and a highly oxidative region (25); Figure 1] were each mounted in embedding medium on a cork, prior to being frozen in liquid isopentane prechilled in liquid nitrogen. Note that the coefficient of variation for sarcomere length is approximately 4% in muscle samples prepared in this manner (26). Muscles were stored at -70°C until processed for histochemistry.

View larger version (22K): [in this window] [in a new window]   Figure 1. Schematic depiction of the white glycolytic region of gastrocnemius muscle (Gw) and the red oxidative region of gastrocnemius muscle (GR). The medial head of the gastrocnemius muscle is identified to provide orientation

Physical Activity Estimation
The amount of voluntary physical activity was estimated in a separate group of 8-month-old (n = 4) and 28–30-month-old (n = 4) F344BN rats. This involved placing individual rats (that had been previously housed two per cage) in a standard cage (21 x 20 x 42 cm) that was resting on two aluminum lever arms instrumented with strain gauges, such that animal movement in the cage was associated with a slight bending of the aluminum lever arms at their fulcrum which, in turn, resulted in a voltage change from the strain gauges. Data from the strain gauges were collected online using a data acquisition unit (Dataq DI-700; Dataq Instruments, Akron, OH) connected to a laptop computer running Windaq Pro+ software (Version 2.39, Dataq Instruments). Data analysis of physical activity recordings involved downloading the raw recorded voltage signal to Matlab (Version 6.5; The MathWorks, Inc., Natick, MA) and integrating the time during which the voltage signal was elevated above a predetermined baseline. Note that the baseline was determined by visually inspecting the behavior of each animal over a 2-hour period and noting the types of movements that yielded a given change in voltage. Those movements that involved only a shift in body weight were omitted from the analysis using a filter set to exclude voltages less than or equal to this baseline voltage (J. L. Hagen, personal communication). Note that the initial 2 hours of data collection for the 72-hour period were discarded from all animals, as this period may not represent the habitual level of voluntary physical activity because of exploratory behavior associated with transfer to a new environment (32).

Statistics
Values were expressed as means ± SE (standard error). Differences between groups were assessed by two-way analysis of variance (ANOVA) (muscle x age for muscle mass, fiber mean cross-sectional area, coefficient of variation [CV%] for fiber cross-sectional area, fiber shape), with a Bonferroni multiple comparison test; or by Student's t test (physical activity, body mass). Comparisons between gastrocnemius and soleus muscles in the late middle-aged group were made using two-way repeated measures ANOVA (muscle x staining pattern for fiber shape and fiber cross-sectional area), with a Bonferroni multiple comparison test. Differences between gastrocnemius and soleus muscles for the percentage exhibiting a given staining pattern were assessed by a Mann-Whitney rank sum test. Differences between normal fibers and those lacking cytochrome oxidase staining for fiber size and shape in the soleus muscles of late middle-aged animals were assessed by paired t test. Frequency histograms were produced for each muscle using Sigmaplot (Version 8.02, SPSS, Inc., Chicago, IL) and the distributions analyzed for normality using a Kolmogorov-Smirnov test. The was set at.05.

	RESULTS

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Animal Characteristics and Voluntary Physical Activity
Body mass and muscle mass data are found in Table 1. The body mass of the late middle-aged animals was significantly greater than the young adult rats. The mass of the triceps surae muscle group (gastrocnemius, plantaris, and soleus muscles) was reduced by approximately 9% in the late middle-aged animals. Whereas the mass of the gastrocnemius muscle was reduced, the mass of the soleus muscle was not different in the late middle-aged animals. Notwithstanding this point, the quotient of body mass and gastrocnemius muscle or soleus muscle mass were both reduced in the late middle-aged animals. The duration of physical activity over a 70-hour period in a separate group of F344BN rats was 34% lower (p <.05) in late middle-aged animals (2.5 ± 0.2 hours; n = 4) than young adult animals (3.6 ± 0.3 hours; n = 4).

View larger version (26K): [in this window] [in a new window]   Figure 2. Frequency histograms of fiber size in soleus muscle (A: 168 ± 17 fibers measured per muscle) and whole gastrocnemius muscles (B: 270 ± 14 fibers measured per muscle) in young adult (8-month-old) and late middle-aged (28–30-month-old) rats. Values are means ± SE (standard error)

View larger version (137K): [in this window] [in a new window]   Figure 3. Light micrographs of soleus (A and B) and gastrocnemius (C and D) muscle from late middle-aged (28–30-month-old) animals stained for lead ATPase (A and C) or succinate dehydrogenase (B and D). Note the marked reduction in subsarcolemmal succinate dehydrogenase staining in the atrophied angular fibers. Numbers identify the same fibers in serial sections. Bar = 50 µm. ATP = adenosine triphosphate

Further inspection of the hypertrophied fibers in soleus muscle of the late middle-aged animals revealed markedly reduced or deficient cytochrome oxidase staining in some of these fibers (Figure 4), whereas succinate dehydrogenase staining was not different from the surrounding nonhypertrophied fibers (data not shown). Note that this staining pattern (normal succinate dehydrogenase, deficient cytochrome oxidase staining) has been described previously as one phenotype indicative of electron transport chain dysfunction consequent to mitochondrial DNA damage (21,33,34), suggesting that some of the hypertrophied fibers may be undergoing pathological changes.

View larger version (54K): [in this window] [in a new window]   Figure 4. Serial sections of muscle stained for lead ATPase (A) or cytochrome oxidase (B) demonstrating the appearance of cytochrome oxidase deficiency in hypertrophied fibers (fiber cross-sectional area >12,000 µm2 for the fiber depicted with an asterisk [*]) in soleus muscle of a late middle-aged (28–30-month-old) animal. Numbers identify the same fibers in serial sections. Bar = 50 µm. ATP = adenosine triphosphate

	DISCUSSION

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The decline in skeletal muscle mass and contractile performance with aging has been attributed to both fiber atrophy and fiber loss. We hypothesized that the nature and causes of these changes with aging would differ between muscles that have a different function. Specifically, we hypothesized that a postural muscle, soleus, would exhibit a better preservation of muscle mass with aging (due to continued activation) and a more heterogeneous fiber size distribution (due to atrophy of some fibers and compensatory hypertrophy in other fibers). On the other hand, we hypothesized that gastrocnemius muscle would demonstrate a reduced mean fiber size, due primarily to reduced activation consequent to lower physical activity levels with aging. Our results supported these hypotheses in that the soleus demonstrated no change in muscle mass, and frequency histograms revealed both severely atrophied (fiber cross-sectional area 1000 µm²) and hypertrophied fibers (8000 µm²) in the late middle-aged animals. Furthermore, consistent with the effects of reduced muscle activation consequent to a 34% lower amount of voluntary physical activity observed in a separate group of late middle-aged animals, the whole gastrocnemius muscle demonstrated a reduction in mean fiber cross-sectional area, but regional analyses showed that, whereas the white (glycolytic) region of gastrocnemius had smaller fibers (as would be expected with lower levels of physical activity), the red (oxidative) region of gastrocnemius had larger fibers relative to that seen in young adult animals. Notwithstanding this point, frequency histograms in the whole gastrocnemius muscle of the late middle-aged animals revealed a similar proportion of severely atrophied fibers to that seen in soleus muscle, and no abnormally hypertrophied fibers relative to that seen in young adult muscles. Interestingly, most of the atrophied fibers in soleus muscle, but fewer of those in gastrocnemius muscle, demonstrated a lack of subsarcolemmal staining for succinate dehydrogenase and cytochrome oxidase, suggesting a preferential loss of subsarcolemmal mitochondria consequent to denervation in these fibers. In contrast, some of the hypertrophied fibers in soleus muscle demonstrated markedly reduced or deficient cytochrome oxidase staining but relatively normal succinate dehydrogenase staining, suggesting mitochondrial dysfunction in these fibers.

Significance of Decreased Subsarcolemmal Mitochondrial Staining
As noted above, we observed that some fibers in both soleus and gastrocnemius muscles of the late middle-aged animals demonstrated a marked reduction in subsarcolemmal succinate dehydrogenase staining. Note that in all instances examined, fibers that exhibited a lack of succinate dehydrogenase staining also exhibited a lack of cytochrome oxidase staining in serial sections (R.T. Hepple and A. B. Rempfer, unpublished observations). We have interpreted this staining pattern as evidence of a preferential loss of subsarcolemmal mitochondria, consequent to denervation, based on the following rationale. Selective myocyte denervation would result in an inability to activate the affected fibers, which could cause a preferential loss of subsarcolemmal mitochondria secondary to a reduced adenosine triphosphate (ATP) demand to drive the subsarcolemmal Na⁺-K⁺ ATPase pumps, consequent to the cessation of membrane depolarization that would occur under these conditions. Consistent with this idea, previous results have shown that muscle inactivity induced by hindlimb suspension (16) or space flight (35) in rodents results in a relatively greater reduction in subsarcolemmal mitochondrial volume than intermyofibrillar mitochondrial volume. More direct support is provided in a study by Borisov and colleagues showing that surgical denervation of whole muscles in rats results in a relatively greater loss of subsarcolemmal mitochondria (36). Note also that the proportion of fibers that were denervated in the soleus muscles (10% ± 4%) and gastrocnemius (8% ± 2%) muscles of the late middle-aged animals, using a lack of subsarcolemmal mitochondria as the criteria in the current study, is similar to that reported previously using antibodies to neural cell adhesion molecule in the extensor digitorum longus of 24-month-old Fischer 344 rats (8% ± 2%) (37). These fibers were also more angular, which has been suggested previously to be indicative of denervation (38). Note that we do not consider a preferential loss of subsarcolemmal mitochondria to be an indication of mitochondrial pathology.

Whole-Muscle Mass and Aging
A decline in skeletal muscle mass is one of the most well-established features of aging. There have been a variety of animal models used to study aging in skeletal muscle, and most of these are rodents, with rats being the most common. It is noteworthy that, whereas a reduced muscle mass is consistently seen with aging in humans, there has been considerable variability in this response between studies using rats as models. This effect is due in part to the very wide range of ages used to represent "old" or "senescent" animals, and perhaps the confounding influence of pathology in the inbred strains typically studied in the past [e.g., see work by Lipman and colleagues (39)]. More recently, survival curves for specific rodent strains have been used to more accurately describe the relative ages of the animals studied. In this respect, a model of aging recently developed by the NIA, the Fischer 344 x Brown Norway F1-hybrid rat, has been shown to demonstrate progressive skeletal muscle atrophy with aging on a consistent basis (10,11,40,41) and a lower incidence of age-related pathologies (39). This was the strain used for this study. The rats in the current investigation represent young adult (8-month-old) and late middle-aged (28–30-month-old) animals, based on previously published survival curves for this strain (13). Thus, the late middle-aged animals correspond roughly to a human age of 60 years, based on published survival curves from humans (23).

The triceps surae muscle group was reduced by approximately 9% in the late middle-aged animals. This degree of muscle mass reduction is somewhat smaller than would be expected between young adulthood and late middle age in humans [15%, estimated based on changes in whole vastus lateralis muscle cross-sectional area in human cadavers by Lexell (1)], although measurements made in a larger sample of animals in our laboratory (see below) reveals a very similar 17% decline across this age range (R. T. Hepple, unpublished). Previous studies in aging rodents have noted that the postural soleus muscle exhibits less whole-muscle atrophy than do locomotor muscles, such as the gastrocnemius muscle [e.g., (11,42)]. The current results are similar in this regard. Note also that, whereas we reported previously that soleus muscle mass was reduced in 28–30-month-old F344BN rats (12), the current results showing no difference in soleus muscle mass between 8-month-old rats and 28–30-month-old rats are consistent with measurements made in a larger number of animals in our laboratory [n = 17 in each group, which includes the animals from our previous study, the current results, and 6 additional animal at each age; R.T. Hepple, unpublished observations].

Factors Contributing to Greater Heterogeneity of Fiber Size With Aging
Similar to the heterogeneity in degree of whole-muscle atrophy, alterations in fiber size are also variable with aging. This heterogeneity reflects an increasing diversity of influences in skeletal muscle with aging, which include physical inactivity, denervation (18), mitochondrial dysfunction (21,22), and apoptosis (43). In this respect, heterogeneous alterations in fiber size with aging would be expected not only when comparing muscles of different function (i.e., postural versus locomotor), but also within individual muscles. Specifically, reduced physical activity will not impact all muscles equally with aging because the postural muscles will experience less reduction in activation compared to the locomotor muscles. Furthermore, one would expect greater heterogeneity in fiber size in muscles that have a relatively maintained activation with aging, due to the opposing influence of processes causing fiber atrophy and death, such as denervation and mitochondrial dysfunction, versus a relative overload of the unaffected muscle fibers (resulting in compensatory hypertrophy). In this respect, the distal hindlimb muscles of the rat possess distinct regions comprised primarily of slow oxidative (soleus muscle), fast glycolytic (white region of gastrocnemius muscle), and highly oxidative (red region of gastrocnemius muscle) muscle fibers (25), and, therefore, can provide significant insight into the functional distribution of alterations in skeletal muscle morphology with aging (see below).

In the current study, we saw a trend to larger mean fiber size in soleus muscle of late middle-aged animals. Since the soleus muscle is postural and thus would experience similar levels of recruitment with advancing age, the tendency to larger fibers is likely explained by the reduction in soleus muscle mass relative to body mass (due exclusively to a higher body mass in the late middle-aged animals in this study), which results in a relative overload with aging compared with young adult animals. Given that the white region of gastrocnemius muscle is primarily glycolytic in nature (25), and thus recruited primarily during more intense types of physical activity (44), the decrease in mean fiber size in this region is likely due to the reduced amount of voluntary physical activity observed in late middle-aged animals. On the other hand, the highly oxidative red region of gastrocnemius muscle (25) might assist the soleus muscle in maintaining posture during stance, and thus, like soleus, may become relatively overloaded compared with young adult animals because of a lower muscle mass to body mass ratio. This would be consistent with the increased fiber size in the red region of gastrocnemius of the late middle-aged animals.

Although the vast majority of previous studies have made age comparisons based on mean fiber cross-sectional area, a number of studies have commented on an increasing heterogeneity of fiber size within muscles with aging. Consistent with previous studies (3,4,45,46), we observed a marked increase in heterogeneity of fiber size in frequency histograms and in comparing the CV% for fiber cross-sectional area in late middle-aged versus young adult muscles, although this was only significant in the soleus muscle. Underscoring this point, we observed both severely atrophied (fiber cross-sectional area area 1000 µm²) and hypertrophied fibers (8000 µm²) in the soleus muscle of late middle-aged animals, whereas gastrocnemius muscle was characterized mainly by a shift toward smaller fibers in the late middle-aged animals. Since the soleus muscle is likely recruited to a similar extent with aging, as argued above, the divergence in fiber size likely reflects competing influences of aging-associated processes versus relative overload of the soleus muscle. In the former respect, most of the fibers 1000 µm² in soleus muscles and about one third of these fibers in the gastrocnemius muscles of the late middle-aged animals demonstrated a phenotype consistent with the influence of denervation (reduced subsarcolemmal mitochondrial staining). On the other hand, none of the atrophied fibers in soleus muscles demonstrated cytochrome oxidase deficiency in the interior (cytoplasm) of the fibers. Since this phenotype (cytochrome oxidase deficiency) has been shown to coincide with mitochondrial DNA damage and on this basis has been interpreted as evidence of mitochondrial dysfunction (21), our results suggest that mitochondrial dysfunction did not contribute significantly to the severe atrophy observed in the soleus muscle. In contrast, we also observed evidence of selective fiber hypertrophy in the soleus muscle with aging. Although we believe this to be a physiological response to a relative overload of these fibers in reaction to selective fiber atrophy in other fibers coupled with a modest increase in body mass, some of the hypertrophied fibers demonstrated very low or deficient cytochrome oxidase activity, suggesting mitochondrial dysfunction in some of these fibers. Note that this characteristic was particularly evident in one muscle out of the three examined in the late middle-aged animals, and this animal also exhibited the most severe fiber hypertrophy of those examined. Given the extreme size of some of these fibers (exceeding 12,000 µm² in some cases), we speculate that reduced O₂ levels in the core of these fibers may lead to a corresponding increase in free radical generation [as seen in hypoxia; for a review see (47)], accelerating the age-related damage of mitochondrial DNA, and lead to the characteristic cytochrome oxidase deficiency often seen in muscle fibers afflicted in this manner [e.g., (21)]. Since recent studies have linked cytochrome oxidase deficiency to fiber atrophy and breakage (21,22), excessive compensatory hypertrophy in some aging muscles could prove to be another important contributor to fiber death and, thus, sarcopenia with aging.

Summary
Our results demonstrate a marked heterogeneity of alterations in fiber size within and between individual skeletal muscles with aging. In particular, whereas mean fiber cross-sectional area in late middle-aged animals was reduced in the gastrocnemius muscle as a whole and in the white region of this muscle, the red region of gastrocnemius exhibited increased mean fiber size. Similarly, soleus muscles of late middle-aged animals demonstrated a trend to greater fiber size. Frequency histograms underscored this increasing heterogeneity with aging in that they demonstrated the presence of both atrophied (fiber cross-sectional area 1000 µm²) and hypertrophied (8000 µm²) fibers in soleus muscles, and atrophied fibers in gastrocnemius muscles of late middle-aged animals, relative to that seen in young adult animals. Interestingly, most of the atrophied fibers in soleus muscles exhibited a loss of subsarcolemmal mitochondrial staining, consistent with the effects of denervation, whereas some of the severely hypertrophied fibers in soleus muscles exhibited deficient cytochrome oxidase staining, suggesting pathological mitochondrial alterations in these fibers. These findings underscore the divergent influences (e.g., physical inactivity, denervation, mitochondrial dysfunction) acting on skeletal muscles with aging. As a closing point, in the future it would be interesting to determine whether there is a fiber-type specificity to any of these responses.

	Acknowledgments

Address correspondence to Russell T. Hepple, PhD, Faculty of Kinesiology, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4. E-mail: hepple{at}ucalgary.ca

	Footnotes

 
Decision Editor: James R. Smith, PhD

Received July 18, 2003

Accepted November 10, 2003

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