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Correspondence to: John P. Mattson, ATC 1850 East 250 South, Room 241, Dept. of Exercise and Sport Science, University of Utah, Salt Lake City, UT 84112-0920. E-mail: jmattson@hsc.utah.edu
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Summary |
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The hamster is a valuable biological model for physiological investigation. Despite the obvious importance of the integration of cardiorespiratory and muscular system function, little information is available regarding hamster muscle fiber type and oxidative capacity, both of which are key determinants of muscle function. The purpose of this investigation was to measure immunohistochemically the relative composition and size of muscle fibers composed of types I, IIA, IIX, and IIB fibers in hamster skeletal muscle. The oxidative capacity of each muscle was also assessed by measuring citrate synthase activity. Twenty-eight hindlimb, respiratory, and facial muscles or muscle parts from adult (144147 g bw) male Syrian golden hamsters (n=3) were dissected bilaterally, weighed, and frozen for immunohistochemical and biochemical analysis. Combining data from all 28 muscles analyzed, type I fibers made up 5% of the muscle mass, type IIA fibers 16%, type IIX fibers 39%, and type IIB fibers 40%. Mean fiber cross-sectional area across muscles was 1665 ± 328 µm2 for type I fibers, 1900 ± 417 µm2 for type IIA fibers, 3230 ± 784 µm2 for type IIX fibers, and 4171 ± 864 µm2 for type IIB fibers. Citrate synthase activity was most closely related to the population of type IIA fibers (r=0.68, p<0.0001) and was in the rank order of type IIA > I > IIX > IIB. These data demonstrate that hamster skeletal muscle is predominantly composed of type IIB and IIX fibers. (J Histochem Cytochem 50:16851692, 2002)
Key Words: skeletal muscle, muscle fiber type, myosin heavy chain
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Introduction |
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THE HAMSTER constitutes a unique and important biological model for investigation of physiological function and pathological dysfunction. Specifically, since 1963 more than 79,000 articles have been published (PubMed Database) with use of or reference to the hamster. The hamster has been utilized to investigate pathological dysfunction stemming from diabetes mellitus (
Hamster skeletal muscle fiber classification has been reported for a limited number of muscles, such as the cremaster (
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Materials and Methods |
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The protocols used in this investigation were approved by the University of Utah Institutional Animal Care and Use Committee. In all respects, they conform with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 8523, revised 1985).
Animals and Muscles
Three adult male Syrian golden hamsters (78 months old, 146 ± 2 g bw) were maintained on a 12:12-hr light:dark cycle and were supplied with rodent chow and water ad libitum.
Hamsters were weighed and sacrificed with ketamine/xylazine. Major muscles from the face, thorax, hip, thigh, and leg were dissected bilaterally and weighed. Popesko and colleagues' color atlas (
After being weighed, a 5-mm midbelly portion was dissected from one set of muscles for each animal, frozen in melting isopentane, and stored at -70C for immunohistochemical determination of fiber composition and cross-sectional area. The second set of muscles were frozen in liquid N2 and stored at -70C for determination of citrate synthase activity.
Citrate Synthase Activity
Activity of citrate synthase, a mitochondrial enzyme and marker of muscle oxidative capacity, was measured in frozen muscle samples according to the methods described by
Immunohistochemical Analysis
Serial transverse cross-sections (810 µm) near the midbelly portion of each muscle were cut on a cryostat microtome. Fiber type identification was performed as described by
Determination of Muscle Fiber Composition and Cross-sectional Area
All the fibers contained in each muscle cross-section were typed to determine the relative population of each fiber type. For quantification of fiber cross-sectional area, muscle cross-sections were divided into four or five evenly spaced non-overlapping regions, depending on the size of the sample. Representative fascicles with fibers cut perpendicular to their long axes were measured with the use of an image processing system (Bioquant; Nashville, TN). A minimum of five fibers of each type was measured in each of the four or five regions of the muscle. Therefore, in every muscle, fiber area for each of the fiber types was measured in 2040 fibers. Exceptions to this procedure were made when only a few fibers of a given fiber type were present in a muscle. Under this circumstance, fiber cross-sectional area was measured in all the fibers present of that type in the muscle cross-section. Similar sampling techniques have been previously used to determine fiber population and area (
Estimation of Fiber Mass
For each muscle, the relative portion of each fiber type was estimated as previously described (
where f is type I, IIA, IIX, or IIB.
To compute the mass of the muscle composed of each of the four fiber types, it was assumed that fiber mass makes up 85% of the total muscle mass (Mt) (
This computation also assumes that the density and length of the fibers are not significantly different among types.
Statistical Analysis
Differences between cross-sectional areas among different fiber types were determined by calculating a 95% coefficient interval around sample means. Linear regression analysis was used to determine the relationship between oxidative potential (citrate synthase) and the percentage of each fiber type.
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Results |
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Muscle fiber composition and mass data are presented in Table 1. Muscles analyzed ranged in size from soleus (22 ± 3 mg) to biceps femoris muscle (1285 ± 69 mg). As in the rat (
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Mean cross-sectional area of type I fibers ranged from 922 ± 69 µm2 in extensor digitorum longus to 2618 ± 580 µm2 in pectineus muscles (Table 1). Mean cross-sectional area of type IIA fibers ranged from 1175 ± 308 µm2 in soleus to 3172 ± 178 µm2 in cheek pouch retractor muscles. Mean cross-sectional area of type IIX fibers ranged from 1634 ± 683 µm2 in cheek pouch to 5367 ± 2264 µm2 in the caudal portion of adductor muscles. Finally, mean cross-sectional area of type IIB fibers ranged from 2273 ± 405 µm2 in the lateral portion of gastrocnemius to 6067 ± 790 µm2 in gracilis muscles. Mean fiber cross-sectional area across all muscles was 1665 ± 328 µm2 for type I fibers, 1900 ± 417 µm2 for type IIA fibers, 3230 ± 784 µm2 for type IIX fibers, and 4171 ± 864 µm2 for type IIB fibers (Table 2).
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Soleus muscle was the only muscle or muscle part sampled in which type I fibers constituted the majority of fibers in the muscle. Type I fibers also made up the largest portion of muscle mass in the soleus muscle. In eight of the 28 muscles or muscle parts, type IIA fibers constituted the highest portion of fiber type percentage. In only four of these muscles was the largest portion of muscle mass made up of type IIA fibers. In 12 of the 28 muscles or muscle parts, type IIX fibers constituted the highest portion of fiber type percentage and, in 15 of the 28 muscles, type IIX fibers made up the largest portion of muscle mass. In six of the 28 muscles or muscle parts, type IIB fibers constituted the highest portion of fiber type percentage and, in seven of the 28 muscles, type IIB fibers made up the largest portion of muscle mass. The remaining muscle, pectineus, was principally composed of equivalent amounts of type I and IIA fibers. Collectively, type IIX fibers comprised 39% of the total mass of the muscles sampled, whereas type IIB fibers made up the greatest portion (40%) of the muscle mass (Table 2). Type IIA fibers constituted 16% of the total muscle mass and type I fibers made up 5%.
Citrate synthase activity ranged from 9.9 µmol/min/g wet wt in cheek pouch muscle to 64.6 µmol/min/g wet wt in costal portion of diaphragm muscle (Table 1). The strongest correlation between citrate synthase activity and a single fiber type percentage was with type IIA fibers (r=0.68; Table 3) and inversely with type IIB fibers (r=-0.55). However, the strongest relationships between fiber composition and oxidative capacity occurred when type I and IIA fibers were grouped (r=0.71) and inversely when type IIX and IIB fibers were grouped (r=-0.69).
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Discussion |
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The primary purpose of this investigation was to measure immunohistochemically the relative composition and size of muscle fibers composed of types I, IIA, IIX, and IIB fibers in hamster skeletal muscle and to determine the muscle oxidative capacity, as indicated by citrate synthase activity. An understanding of fiber composition, fiber-specific muscle mass, muscle mass, or muscle group mass, and oxidative potential is useful for investigations integrating cardiorespiratory and muscular systems that examine physiological function and/or pathological dysfunction. For example, many investigations, through necessity, use a very specialized hamster muscle (e.g., check pouch retractor for intravital microscopy), and data derived from such unique muscles should ideally be interpreted in the context of their fiber composition and oxidative capacity when relevant.
Previous work characterizing muscle fiber composition in the hamster musculature was based on myosin ATPase and mitochondrial NADH-TR activity (
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As described for rat skeletal muscle, mean fiber cross-sectional area of hamster skeletal muscle varies among fiber types. Specifically, the smallest fibers are type I fibers, the largest are type IIB fibers, and type IIX are intermediate to type IIA and IIB fibers (Table 2). In hamster hindlimb musculature, type IIA fibers demonstrated the least heterogeneity with a 2.2-fold difference in fiber cross-sectional area across muscles. Type I, IIX, and IIB fibers all had similar differences in fiber size across muscles (2.8, 2.8, and 2.7-fold difference in cross-sectional area, respectively). In addition, for a given fiber type there is a spatial distribution in fiber size among muscles, as has previously been described in other animal species (
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In the present study, correlational analysis indicated that the oxidative potential of hamster muscle is greatest in muscles composed primarily of type IIA fibers, and in the rank order of type IIA > I > IIX > IIB fibers (Table 3). However, unlike the fiber classification scheme of
The rank order of fiber type and oxidative potential in hamster muscle is identical to that reported for the rat using similar correlational analysis and histological determination of fibers and oxidative potential (
In summary, the results of the present investigation demonstrate that the relative mass of hamster skeletal muscle is predominantly composed of type IIX (39%) and type IIB (40%) fibers. In addition, there is a continuum of type IIX fiber composition among muscles, ranging from 0% in soleus to 100% in the cheek pouch muscle. Activity of citrate synthase, a marker of muscle oxidative capacity, was strongly correlated with the population of type IIA fibers and, similar to the rat, fell in rank order of type IIA > I > IIX > IIB. Muscle fiber cross-sectional area and distribution also varied within and among muscles in the hamster. The mean cross-sectional area of type IIX fibers was intermediate to type IIA and IIB fibers and fell in rank order of type IIB > IIX > IIA > I. Type I and IIA fibers tended to be more concentrated in deep limb muscles and deep portions of muscles, whereas type IIX and IIB fibers tended to be most concentrated in superficial muscles and muscle parts (Fig 2).
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Acknowledgments |
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Supported by an American Lung Association grant RG-013-N, an American College of Sports Medicine Visiting Scholar Award, and a National Heart, Lung, and Blood Institute grant HL-50306.
We gratefully acknowledge Timothy I. Musch, PhD, and Sue Hageman for technical assistance with this project.
Received for publication March 6, 2002; accepted June 26, 2002.
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