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1YS New Technology Institute, Tochigi 329-0512; 2Laboratory of Reproductive Physiology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502; 3Department of Laboratory Animal Science, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613; 4Department of Life Sciences, The Graduate School of Arts and Sciences, The University of Tokyo, Tokyo 153-8902; and 5Developmental Biology Department, Development and Differentiation Laboratory, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan
Submitted 15 October 2002 ; accepted in final form 7 April 2003
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ABSTRACT |
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genetic background; maximal running time; growth; Mus spretus
It is possible to evaluate a mouse's physical performance by using an exercise test on a treadmill (7, 28). Such physical performance tests are an effective measure of physiological ability. However, the tests have had difficulty in demonstrating large strain differences between classical laboratory inbred mice. Recently, Lerman et al. (17) reported that the forced endurance exercise performance of FVB/NJ (FVB) is very high in the classical laboratory mouse strains. Lerman's report is a worthy contribution to experimental animal models of exercise physiology science. However, the main genetic background of old inbred strains was derived from a European subspecies of M. musculus, Mus musculus domesticus (34, 35, 37). Therefore, we tried to use newly established strains from wild mice, as reported by Koide et al. (14). In general, physical performance could be influenced by various physiological (respiratory-circulatory function, energy metabolism, musculo-skeletal function, and neural transmission) and psychological factors (emotion, etc.). Moreover, the relative contributions of those functions to physical performance could be changed by the introduction of a different genetic background. Garland et al. (8) addressed such a question by an intraspecific comparison of the laboratory mouse and the wild mouse (M. m. domesticus). They examined whether differences in sprint speed in an individual mouse are correlated with gastrocnemius muscle mass or the percentage of cross-sectional area occupied by each major skeletal muscle fiber type. Their results suggested a lack of correlation between sprint speed ability and skeletal muscle size or fiber composition. Use of wild-derived inbred mice of subspecies or species closely related to the classical laboratory mouse strains might reveal genetic factors underlying sprint speed ability. Moreover, skeletal muscle, which is a major organ for locomotor activity, has a species-specific fiber type (26, 33). Therefore, we compared skeletal muscle traits in wild-derived inbred mice with those in an existing inbred strain. Previous reports regarding not only skeletal muscle but also respiratory-circulatory function have shown evidence of genetic influences. For example, Bouchard et al. (3) showed a significant genetic effect on physical performance in a human twin study.
In the present study, we compared basic data on growth, tissue weights, maximal running time, and features of the soleus muscle in M. m. domesticus (B6 and BALB), Mus musculus molossinus (MSM/Ms), and M. spretus. Our results demonstrate that the wild-derived inbred mouse strains are an effective model for enlarging the variability in physical performance phenotypes and for studying the relationship between such phenotypic characteristics and genetic factors.
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METHODS |
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Growth curve and anatomic data. All male mice were measured to determine the change in body weight from 3 to 15 wk of age. The results were compared among the four mouse strains (B6, BALB, MSM/Ms, and M. spretus) to determine the effect of genetic background on growth rate and body size. The growth rate was calculated as (body wt of the present week - body wt of the previous week)/body wt of the previous week x 100. At 12 wk of age, the mice were euthanized after weighing, and the weights of the heart, liver, and abdominal (mesenterical, retroperitoneal, and epididymal) adipose tissue and tail length were recorded (5, 16).
Muscle fiber typing and the cross-sectional areas of soleus. The fiber types of soleus muscles from B6, BALB, MSM/Ms, and M. spretus were analyzed at 12 wk of age. A small block of soleus muscle was frozen in isopentane cooled in liquid nitrogen. The frozen muscles were serially cryosectioned to a thickness of 7 µm at -20°C. Individual fibers in the soleus were histochemically classified as type I, IIa, IIb, and IIc fibers by myosin ATPase activity (4, 10, 25). Samples were preincubated for a total of 30 min in sodium barbital buffer (0.03 M) at the following pH levels: 4.0, 4.2, and 4.4 (5 min each), 10.3 (15 min). After the enzyme reaction, we measured the total fiber numbers, and the composition and area of each fiber type group in the soleus muscles of all four inbred mouse strains (4, 25). The cross-sectional area of each fiber type per muscle was analyzed by using National Institutes of Health Image software.
Physical performance test. Maximal running time until exhaustion, with the use of an incremental protocol, was measured as an index of physical performance among the four strains at 10 wk of age (11, 24). All mice were adapted to the treadmill for a period of 10 days. During the adaptation period, the mice were made to run on the treadmill for 10 min at a speed of 20 m/min. After treadmill practice, each mouse was carried to the grid and trained to associate the grid with an electrical shock. Each mouse was then placed in a small chamber equipped with an electric shock grid that was held on the treadmill belt (Clea Japan, Tokyo, Japan). Running speed was gradually increased by 10 m/min every 1 min from an initial speed of 20 m/min, with a constant grade of 0%, until the individual could not keep running on the moving belt. Exhaustion was defined as the point at which the mouse could no longer run on the treadmill to avoid a shock from the grid. In a preliminary experiment using three males of each mouse strain, we repeated this physical performance test on multiple days. This experiment demonstrated that the results for individual mice across trials were nearly identical (data not shown). Food was taken away from the cage 6 h before the test. The test was performed from 1300 to 1500 (i.e., during the dark cycle) to maximize physical performance. The ambient temperature during the test was kept at 23 ± 3°C.
Statistical analysis. All data are reported as means ± SE. Body weight data, organ indexes (which are indicated as organ weight/body wt x 100), and running time until exhaustion were statistically compared across strains by a one-way ANOVA followed by a Fischer's paired least-significant difference. We also tested whether the running times until exhaustion of individual mice were correlated with body weight and characteristics of soleus muscle. All statistical analyses were performed by using Stat View.
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RESULTS |
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7 wk of age).
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Tissue weights and tail length at 12 wk of age. Mean tissue weights of all strains at 12 wk of age are shown in Table 1. Organ indexes (organ weight/body weight x 100) of heart, pancreas, kidney, and abdominal adipose tissues, also shown in Table 1, differed significantly (P < 0.05) among the four strains. In particular, the organ index of heart of M. spretus was the heaviest, whereas the pancreas index of M. spretus was the lightest among all mouse strains. The tail length, which is one of skeletal size (5, 16), at 12 wk of age also differed significantly (P < 0.05) among the four inbred strains.
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Total fiber number, type, and the cross-sectional areas of soleus muscle. There are significant differences in the fiber number of soleus muscle between the two wild-inbred mouse strains, M. m. molossinus and M. spretu (P < 0.05), with that of M. spretus being approximately one-half that of M. m. molossinus and onethird that of M. m. domesticus (Table 2). The soleus muscle fiber types differed significantly (P < 0.05) at 12 wk of age among M. m. domesticus, M. m. molossinus, and M. spretus (Table 2 and Fig. 2). The main fiber types in the soleus muscle were type I for M. spretus, whereas type IIa dominated for the classical laboratory strains. No difference was observed within species. The soleus muscle of M. m. molossinus consisted of almost equivalent percentages of type I and type IIa. Type IIb and IIc could not be recognized in the soleus muscle of M. spretus. The cross-sectional area of type I of M. spretus was significantly larger than the area of the other three mouse strains (Table 2 and Fig. 2). There is not a significant difference in all mouse strains at the each subtype in type II.
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Relationship between treadmill exercise performance and body weight or characteristics of soleus muscle in wild-derived and laboratory mouse strains. Maximal running time was compared across strains at 10 wk of age (Fig. 3). The exercise performance time was significantly different (P < 0.05) between subspecies and/or interspecies, i.e., subspecies of the laboratory mouse showed higher performance levels than those of the other three inbred mouse strains. On the other hand, running time until exhaustion of M. spretus, a closely related species to the laboratory mouse, was shorter than the running time of the other three inbred mouse strains. The maximal running speed at exhaustion was 60–70 m/min for B6 and BALB/c, 70–90 m/min for MSM/Ms, and 40–60 m/min for M. spretus.
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The correlation of the maximal running time with body weight was compared because there are great differences in body weight among the strains. There was indeed a significant correlation between weight and running time (r = -0.31, P < 0.05) in all mice without considering the genetic background (Fig. 4A). However, running time was not significantly correlated with body weight within any mouse strains (B6, r = -0.24; BALB/c, r = -0.48; MSM/Ms, r = 0.50; and M. spretus, r = 0.23; all P > 0.05). The correlations of the maximal running time with total fiber number (Fig. 4B), proportion of type I (Fig. 4C) and IIa (Fig. 4D), and the cross-sectional areas of type I (Fig. 4E) and IIa (Fig. 4F) were compared. There was indeed a significant correlation between the running time and proportion of type I (r = -0.47, P < 0.05), proportion of type IIa (r = 0.57, P < 0.05), and cross-sectional areas of type I (r = -0.68, P < 0.05) in all mice without considering the genetic background. However, the running time was not significantly correlated with body weight within any mouse strains.
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DISCUSSION |
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Growth rate and anatomic characteristics. The growth pattern of the classical laboratory mouse is generally divided into four growth phases (29): phase 1 is postembryonic development, phase 2 is the highest growth period preceding sexual maturation, phase 3 comprises skeletal maturation, and phase 4 constitutes the completion of maturation. A difference in body weight between the classical laboratory and wild-derived inbred mouse strains was clearly observed after weaning and continued until 15 wk of age (Fig. 1). In particular, the growth rate of M. spretus appeared to differ from the other inbred strains at phase 2. In addition to growth differences, we also observed differences in some anatomic characteristics. For example, several significant differences in organ indexes were found. The organ index of the pancreas was significantly smaller in M. spretus than in the other three inbred mouse strains, which suggests differential glucose metabolism (P < 0.05; Table 1). The organ index of the heart also differed significantly from that of the classical laboratory mouse strains (P < 0.05; Table 1), suggesting a difference in circulatory function between laboratory and wild-derived inbred mouse strains, especially M. spretus (P < 0.05; Table 1). From these results, we predict a large difference in physiological function between wild-derived and laboratory mice.
Characteristics of the soleus muscles of wild-derived mice. We
examined muscle characteristics of the soleus muscle, which is an important
hindlimb muscle because of its participation in the maintenance and regulation
of postural activity (6).
Mammalian skeletal muscles consist of fast- and slow-twitch muscle fibers. The
soleus of the wild-derived inbred mouse strains, especially M.
spretus, at 12 wk of age showed interesting fiber types and
cross-sectional areas (Table 2
and Fig. 2). Unlike the other
three inbred mouse strains, the fibers of the M. spretus soleus
muscle were dominated by type I. Wigston et al.
(32) reported that the
relative proportions of the fibers of mouse soleus staining positively with
fast and slow myosin antibodies were similar at all ages studied, with
60–70% being fast and 30–40% being slow. Then, the type I of
M. spretus had the cross-sectional area of the close double for those
of the other three mouse strains (Table
2). Therefore, most of the cross section of soleus muscle was
closed by the slow-twitch muscle fibers. M. spretus did not have type
IIc fibers that have characteristics intermediate to the slow- and fast-twitch
muscle fiber type (12). The
muscle fiber type of MSM/Ms was also surprising; type I and IIa fibers were
found in almost equal proportions in the soleus muscle of MSM/Ms
(Table 2). The fast- and
slow-twitch muscle fibers seem to equally exist, when cross-sectional areas of
type of I and IIa are considered (Table
2). Thus soleus muscle fiber type of these wild-derived mouse
strains was clearly different from those of the laboratory mouse strains. Not
only did fiber composition and the area differ, but fiber number differed
among groups as well (Table 2).
M. spretus soleus muscle contained the smallest number of fibers of
all mouse strains. Thus the soleus muscles of M. m. domesticus (B6
and BALB), M. m. molossinus (MSM/Ms), and M. spretus
indicated a peculiarity of species. Although determining the complete
relationship between skeletal muscle characteristics and physical performance
from a single skeletal muscle is not possible, our results suggest the
potential utility of wild-derived mouse strains in examining the genetics of
skeletal muscle characteristics.
Treadmill physical performance of wild-derived inbred mouse
strains. Physical performance has been evaluated as running time and/or
speed, sustaining power, and spontaneous activity to examine the
characteristics of wild-derived inbred mice. We compared the running time
until exhaustion of classical laboratory strains at 10 wk of age by using the
maximal exercise test (11,
24) on a treadmill
(Fig. 3). Our results suggest
that this performance test could be an indicator of aerobic exercise capacity
(18) in the mice. Although we
did not measure maximal oxygen consumption
(
O2) directly, the
differences in performance may correlate with differences in maximal
O2, because
O2 linearly increases as
treadmill-running speed increases up to a maximal rate
(27). However, the heart organ
index indicates that the cardiovascular capacity of M. spretus was
the largest of the mouse strains in the present study.
Relationship between physical performance and body weight and
characteristics of soleus muscle in wild-derived and laboratory mouse
strains. It is well known that the prevalence of type I fibers, which
have a high oxidative capacity, is related to high aerobic capacity. However,
in the present study, running time until exhaustion was not related to the
type I fiber ratio of the soleus muscle
(Fig. 4). The running time of
M. spretus, which has soleus muscles mainly composed of type I
(slow-twitch type), was the lowest, whereas that of MSM/Ms, which has soleus
muscles with an almost equal ratio of type I and IIa fibers (fast-twitch
type), was the highest. The maximal exercise test is designed to require the
display of largest power with the endurance ability in each mouse. Therefore,
in the present physical performance test, maximal power would be required for
the mouse. Power might be affected not only by differential
O2 capacity or skeletal
muscle characteristics of each mouse strain but also by communicative
competence of skeletal muscle and the nervous system to adapt running speed.
Furthermore, clear differences in behavioral responses to novelty in an open
field have been reported, with M. spretus being much less active than
B6 (15). From these results,
we propose that the complexity of factors affecting physical performance
necessitates the use of wild-derived strains to study such polygenic traits.
Moreover, the strain dependence of long-time endurance exercise ability raises
an interesting possibility. As endurance runners have a significantly higher
percentage of fiber types I and IIc than nonrunners
(31), M. spretus
should be examined for its endurance running ability.
It is without doubt that the widely used classical laboratory mice represent an important tool for identifying specific genes of interest and their functions. However, introduction of large phenotypic variation into the background of laboratory mice is also useful in precisely analyzing gene functions. For this reason, we hope our study will encourage the use of wild-derived mice for studying the genetic basis of physical performance. In other words, the characteristics of the wild-derived mouse will have better utility for the analysis of complicated genetic traits, such as physical performance and behavior. In conclusion, we have demonstrated a practical system for evaluating physical performance that includes the wild-derived mouse. Moreover, the genetic and environmental factors underlying interesting differences in physical capacity between the wild-derived mouse and the laboratory mouse should be clarified by future work.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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)
and BALB/c (
)] and wild-derived inbred mice [Mus musculus
molossinus (MSM/Ms; 
) and Mus spretus (
)] from 3 to
15 wk of age. The body weights are shown as means ± SE for n =
10–20 mice.
