Fiber Composition and Oxidative Capacity of Hamster Skeletal Muscle

Volume 50(12): 1685 1692, 2002 The Journal of Histochemistry & Cytochemistry http://www.jhc.org ARTICLE Fiber Composition and Oxidative Capacity of Hamster Skeletal Muscle John P. Mattson, Todd A. Miller, David C. Poole, and Michael D. Delp Department of Exercise and Sport Science, University of Utah, Salt Lake City, Utah (JPM); Departments of Health and Kinesiology and Medical Physiology, and the Cardiovascular Research Institute, Texas A&M University, College Station, Texas (TAM,MDD); and Departments of Anatomy, Physiology and Kinesiology, Kansas State University, Manhattan, Kansas (DCP) SUMMARY 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 (144 147 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, type IIA fibers 16%, type IIX fibers 39%, and type IIB fibers 40%. Mean fiber cross-sectional area across muscles was 1665 328 m 2 for type I fibers, 1900 417 m 2 for type IIA fibers, 3230 784 m 2 for type IIX fibers, and 4171 864 m 2 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 50:1685 1692, 2002) KEY WORDS skeletal muscle muscle fiber type myosin heavy chain 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 (Sims and Landau 1967), hypoglycemia (Sodoyez et al. 1969), muscular dystrophy (Lochner et al. 1971), cardiomyopathy (Schwartz et al. 1972), hypothermia (Resch and Musacchia 1976), emphysema (Karlinsky and Snider 1978), skeletal muscle transplantation (Faulkner et al. 1983), age (Nichols and Borer 1987), Correspondence to: John P. Mattson, PhD, 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 Received for publication March 6, 2002; accepted June 26, 2002 (2A5763). chronic wasting infection (Drew et al. 1988), skeletal muscle ischemia/reperfusion (Messina 1990), inactivity (Zhan and Sieck 1992), and gene therapy (Xiao et al. 2000). Moreover, the hamster has been used to explore the physiological function of the myocardium (Aomine et al. 1982), vasculature (Burns and Palade 1968), and skeletal muscle (Lossnitzer and Kelly 1968). In other animal species, investigations integrating the cardiorespiratory and muscular systems have demonstrated the importance of understanding the fiber composition of skeletal muscle. For example, it has been determined that skeletal muscle perfusion rates (Laughlin and Armstrong 1982; Armstrong et al. 1987) and vascular control mechanisms (Laughlin et al. 1989; Schwartz and McKenzie 1990; McCurdy et al. 2000) vary according to muscle fiber composition. In addition, because muscle fiber type and oxidative capacity are key determinants of muscle function in other animal species, knowledge of deviations in these The Histochemical Society, Inc. 0022-1554/02/$3.30 1685

1686 Mattson, Miller, Poole, Delp variables may be important for interpretation of physiological and pathological responses examined in the hamster. Hamster skeletal muscle fiber classification has been reported for a limited number of muscles, such as the cremaster (Sarelius et al. 1983), extensor digitorum longus and soleus (Wilcox et al. 1989), medial gastrocnemius (Zhan and Sieck 1992), and scalene muscles (Fournier and Lewis 2000). In addition, Farkas and Roussos (1984) reported the percentage and crosssectional area of fibers in diaphragm, intercostal, and plantaris muscles. Few studies, however, have reported the oxidative potential of hamster muscle (Farkas and Roussos 1984). Therefore, 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. In addition, the oxidative capacity of each muscle was assessed by measuring citrate synthase activity. Materials and Methods 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 (7 8 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 (Popesko et al. 1990) was used as a reference for the dissection. Larger muscles were not subdivided into red, white, and mixed portions because, unlike the rat (Armstrong and Phelps 1984; Delp and Duan 1996), there appeared to be no gross differences within a muscle. In other words, there were no visually apparent gradations or stratification of color within a given muscle during the dissection. For most muscles, the entire muscle was dissected free and weighed. However, the diaphragm, biceps femoris, adductor, and gastrocnemius muscles were dissected into distinguishable portions on the basis of their anatomy. 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 N 2 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 Reichmann et al. (1985). Briefly, the frozen muscle was pulverized under liquid N 2 and total cell enzymes were extracted by homogenizing the muscle powder in a cold extraction buffer (6.5 g KCl 1.6 g glutathione 0.35 g EDTA 500 ml dh 2 O). Enzyme activities, expressed as mol/min/g wet weight, were measured spectophotometrically in 1 ml assay (0.6 ml 100 mm Tris buffer 0.1 ml 3.0 mm acetyl CoA 0.1 ml 1.0 mm DTNB 0.1 ml diluted homogenate, incubated 7 min, 0.1 ml 5 mm oxaloacetate) mixtures at room temperature (i.e., 25C). Immunohistochemical Analysis Serial transverse cross-sections (8 10 m) near the midbelly portion of each muscle were cut on a cryostat microtome. Fiber type identification was performed as described by Schiaffino et al. (1989). Sections of muscle were fixed with cold AFA fixative (50 ml 37% zinc formalin 370 ml 95% ethanol 25 ml glacial acetic acid) for 5 min. Slides were then hydrated for 10 min in PBS before blocking. Power- Block solution (InnoGenex #BS-1310-25; San Ramon, CA) was added to the sections and incubated for 5 min at RT. After removal of excess blocker, primary antibodies to the MHCs type I (BA-D5), type IIA (SC-71), and type IIB (BF-F3) were added to the appropriate sections and the slides were incubated at 4C overnight in a humid chamber. After incubation, slides underwent two 10-min washes in PBS with gentle rotation. After washing, a biotinylated goat anti-mouse Ig secondary antibody (InnoGenex #AS- 2400-16) was added to the sections for 20 min at RT. Slides were washed as described above and streptavidin alkaline phosphatase conjugate (Inno- Genex #CJ-1002-86) was added to the sections and incubated for 20 min at RT. The conjugate was removed by washing (as in prior steps) and a solution of naphthol phosphate buffer (InnoGenex #BS- 080204) and Fast Red dye (InnoGenex #CH-0802-06) was added to the sections and incubated until adequate color development was observed. Sections were counterstained with Mayer s hematoxylin and mounted with Glycergel (DAKO; Carpinteria, CA). Fibers containing the MHCs of interest expressed a red color after exposure to the immunohistochemical staining procedure. Serial muscle sections were also examined for IIX fibers (i.e., fibers that expressed no staining after exposure to any of the heavy-chain an-

Hamster Skeletal Muscle Fiber Composition 1687 tibodies) and hybrid fibers (i.e., fibers that expressed multiple heavy chains). 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 20 40 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 (Armstrong and Phelps 1984; Delp and Duan 1996). Estimation of Fiber Mass For each muscle, the relative portion of each fiber type was estimated as previously described (Armstrong and Phelps 1984; Delp and Duan 1996). The estimated proportion of each fiber type (P f ) within a given muscle was calculated by dividing the product of the mean cross-sectional area of fibers of that type (A f ) and the population of fibers of that type (% f ) by the sum of the proportional areas of all the fiber types P f (%) = [( A f % f ) { Σ( A f % f )}] 100 where f is type I, IIA, IIX, or IIB. To compute the of the muscle composed of each of the four fiber types, it was assumed that fiber makes up 85% of the total muscle (M t ) (Gollnick et al. 1981). Thus, the fiber of a given type (M f ) was calculated as follows M f = M t 0.85 P f 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. Results Muscle fiber composition and 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 (Delp and Duan 1996), fiber composition varied considerably in the muscles. The muscle with the highest percentage of type I fibers was the soleus muscle (70 13%). Cheek pouch, tensor fasciae latae, cranial portion of the biceps femoris, cranial portion of the adductor, and gracilis muscles had no type I fibers. Muscles with the highest percentage of type IIA fibers were scalene (53 3%), tibialis posterior (51 19%), and plantaris (67 4%). In contrast, cheek pouch muscle had no type IIA fibers. Muscles with the highest percentage of type IIX fibers were the cheek pouch (100%) and tensor fasciae latae (84 7%), whereas soleus muscle had no type IIX fibers. Finally, the muscles or muscle parts with the highest percentage of type IIB fibers were cranial and caudal portions of the biceps femoris (75 18% and 62 22%, respectively), semitendinosus (68 25%), and the cranial portion of adductor (69 2%) muscles. Cheek pouch, cheek pouch retractor, sternohyoid, scalene, diaphragm, vastus intermedius, medial portion of gastrocnemius, tibialis posterior, plantaris, soleus, and flexor halicus longus muscles had no type IIB fibers. Mean cross-sectional area of type I fibers ranged from 922 69 m 2 in extensor digitorum longus to 2618 580 m 2 in pectineus muscles (Table 1). Mean cross-sectional area of type IIA fibers ranged from 1175 308 m 2 in soleus to 3172 178 m 2 in cheek pouch retractor muscles. Mean cross-sectional area of type IIX fibers ranged from 1634 683 m 2 in cheek pouch to 5367 2264 m 2 in the caudal portion of adductor muscles. Finally, mean crosssectional area of type IIB fibers ranged from 2273 405 m 2 in the lateral portion of gastrocnemius to 6067 790 m 2 in gracilis muscles. Mean fiber crosssectional area across all muscles was 1665 328 m 2 for type I fibers, 1900 417 m 2 for type IIA fibers, 3230 784 m 2 for type IIX fibers, and 4171 864 m 2 for type IIB fibers (Table 2). 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 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 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. In six of the 28 muscles or muscle parts, type IIB fibers constituted the

1688 Mattson, Miller, Poole, Delp Table 1 Citrate synthase activity and fiber type composition of major muscles of the hamster ( SD) I IIA IIX IIB Muscle Citrate synthase activity ( mol/min/g) Muscle fiber Population (%) Area ( m 2 ) (%) Population (%) Area ( m 2 ) (%) Population (%) Area ( m 2 ) (%) Population (%) Area ( m 2 ) (%) Facial Cheek pouch 9.9 2.4 491 37 417 32 0 0 0 0 0 0 100 1634 683 417 100 0 0 0 Cheek pouch retractor 12.4 5.0 148 11 126 9 15 5 1578 364 9 8 27 5 3172 178 34 27 58 1 3441 1226 82 65 0 0 0 Respiratory Sternohyoid 38.2 7 189 17 161 14 6 1 1112 155 4 2 43 4 2152 901 47 29 51 4 4319 520 110 69 0 0 0 Scalene 39.3 6.0 85 2 72 2 9 6 1526 48 3 4 53 3 2471 61 30 42 38 9 4372 50 39 54 0 0 0 Diaphragm, costal 64.6 7.9 265 23 225 20 26 4 981 124 35 16 36 9 1177 213 57 25 38 13 2543 462 133 59 0 0 0 Diaphragm, crural 59.5 6.1 183 16 156 14 11 9 1571 233 15 10 31 5 1185 442 33 21 58 14 2064 492 108 69 0 0 0 Hip Pectineus 37.0 7.0 70 36 59 31 41 35 2618 580 23 38 41 31 2752 589 23 39 17 17 3624 1073 12 21 1 2 5278 738 1 2 Tensor fasciae latae 16.2 1.9 89 23 76 20 0 0 0 1 2 1723 863 1 1 84 7 3950 632 63 83 15 5 4245 859 12 16 Biceps femoris, cranial 17.9 2.8 402 111 342 94 0 0 0 9 5 1248 286 11 3 16 22 2323 27 36 10 75 18 4111 261 295 87 Biceps femoris, caudal 19.6 3.5 883 48 751 40 7 4 1427 612 19 3 18 16 2044 424 70 9 13 3 3174 1510 77 10 62 22 5065 941 585 78 Semitendinosus 13.5 3.8 571 16 486 14 2 2 1757 102 3 1 6 4 2143 576 13 3 24 21 4624 605 105 21 68 25 5574 983 365 75 Semimembranosus 25.9 5.6 555 81 472 69 12 6 1901 403 30 6 20 4 2656 337 68 15 22 13 3825 815 109 23 46 7 4426 1849 266 56 Adductor, cranial 20.3 4.5 412 158 350 135 0 0 0 13 3 1407 122 15 4 18 5 3855 563 60 17 69 2 4633 903 276 79 Adductor, caudal 24.7 3.6 499 210 424 179 24 12 2136 762 57 13 29 3 2449 834 82 19 21 26 5367 2264 130 31 26 28 5286 1650 156 37 Caudal abductor 44.6 5.4 63 14 54 12 20 8 1605 226 7 14 37 3 1886 82 16 29 26 9 3090 1044 18 33 17 7 3267 579 13 24 Gracilis 23.5 6.0 516 109 439 92 0 0 0 20 3 2385 1048 40 9 25 12 5851 2472 120 27 55 10 6067 790 279 64 Thigh Rectus femoris 32.7 2.4 325 3 276 2 4 2 2529 464 10 4 34 7 2199 241 70 25 60 7 3264 1028 188 68 2 4 3832 751 9 3 Vastus medialis 40.2 5.0 56 12 48 10 13 16 1895 160 5 10 33 9 1523 206 10 21 50 19 2936 374 30 63 4 5 3408 1119 3 6 Vastus intermedius 50.4 3.7 96 8 82 6 34 27 2372 241 29 35 42 10 2208 419 33 41 24 39 2261 807 20 24 0 0 0 Vastus lateralis 45.6 5.1 187 36 159 30 9 6 2367 1031 12 7 44 11 2165 1131 54 34 39 5 3396 1384 76 47 8 16 3275 464 17 12 Leg Gastrocnemius, medial 40.0 3.5 88 16 75 13 14 2 1421 50 8 11 34 9 1477 130 20 27 52 10 2214 107 46 62 0 0 0 Gastrocnemius, lateral 37.5 3.8 145 7 123 6 16 20 1451 495 14 11 32 10 1644 438 31 25 47 26 2631 678 72 59 5 8 2273 405 6 5 Tibialis anterior 41.2 2.1 132 11 112 9 3 1 1466 392 2 2 30 7 1421 357 19 17 57 11 3083 508 77 69 10 7 3288 1358 13 12 Tibialis posterior 45.5 1.6 42 8 36 7 11 4 1577 267 3 9 51 19 1788 526 17 49 38 23 2057 647 15 42 0 0 0 Plantaris 51.7 6.3 47 9 40 8 12 2 1295 265 3 8 67 4 1821 159 26 65 21 5 2339 297 11 27 0 0 0 Soleus 48.0 7.5 22 3 19 3 70 13 1683 256 14 77 30 13 1175 308 4 23 0 0 0 0 0 0 Extensor digitorum longus 44.0 5.8 33 7 28 6 6 2 922 69 1 3 35 6 1280 118 6 23 52 5 2454 612 18 65 7 10 2710 170 3 9 Flexor halicus longus 36.4 3.6 86 5 73 4 10 6 1096 236 4 6 72 3 1740 275 50 69 18 4 2512 300 18 25 0 0 0

Hamster Skeletal Muscle Fiber Composition 1689 Table 2 Fiber type Muscle fiber cross-sectional area and data a Fiber area ( m 2 ) 95% Coefficient interval, ( m 2 ) Fiber % Total muscle I 1665 328 850 2480 310 5 IIA 1900 417 864 2936 880 16 IIX 3230 784 1282 5178 2191 39 IIB 4171 864 2025 6317 2298 40 a Fiber area values are means SD. Coefficient interval was calculated around fiber area means. Fiber is total summed from all muscles/muscle parts for each fiber type. highest portion of fiber type percentage and, in seven of the 28 muscles, type IIB fibers made up the largest portion of muscle. 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 of the muscles sampled, whereas type IIB fibers made up the greatest portion (40%) of the muscle (Table 2). Type IIA fibers constituted 16% of the total muscle and type I fibers made up 5%. Citrate synthase activity ranged from 9.9 mol/min/g wet wt in cheek pouch muscle to 64.6 mol/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). Discussion The primary purpose of this investigation was to measure immunohistochemically the relative composition Table 3 Linear regression of muscle citrate synthase activity ( mol/min/g) as a function of fiber type percentages of all muscles/muscle parts Fiber type Regression coefficient Intercept p I 0.44 2.66 0.018 IIA 0.68 3.36 0.001 IIX 0.15 46.28 0.444 IIB 0.55 51.21 0.002 I IIA 0.71 0.71 0.001 I IIX 0.16 43.63 0.421 I IIB 0.30 48.56 0.120 IIA IIX 0.34 49.64 0.072 IIA IIB 0.10 54.58 0.628 IIX IIB 0.69 97.50 0.001 I IIA IIX 0.59 46.99 0.001 I IIA IIB 0.19 51.92 0.331 I IIX IIB 0.66 94.84 0.001 IIA IIX IIB 0.34 100.9 0.072 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, muscle, or muscle group, 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 (Farkas and Roussos 1984; Sarelius et al. 1983) or solely on the muscle fibers ATPase activity (Wilcox et al. 1989; Zhan and Sieck 1992). Spurway (1981) suggested there was a close relationship between muscle fiber classification on the basis of ATPase and NADH-TR reactions (Barnard et al. 1971; Peter et al. 1972) and the ATPase reaction alone (Brooke and Kaiser 1970). Thus, type I fibers were purported to be the equivalent of SO fibers, type IIA fibers the equivalent of FOG fibers, and IIB fibers synonymous with FG fibers. As has been previously observed in rat musculature (Delp and Duan 1996), hamster skeletal muscles with a markedly heterogeneous fiber composition do not appear to have percentages of FOG/FG fibers corresponding to those of type IIA/IIB fibers (Figure 1). Farkas and Roussos (Farkas and Roussos 1984), using the method of Peter et al. (Barnard et al. 1971; Peter et al. 1972), classified type IIX fibers as FOG rather than FG fibers using the NADH-TR stain as a result of the greater oxidative capacity of IIX than IIB fibers. Furthermore, using the method of Brooke and Kaiser (1970), Wilcox and colleagues (1989) classified type IIX fibers as type IIB rather than IIA fibers as a result of the similar ATPase staining between IIX and IIB fibers with an acid (ph 4.45) preincubation. The idiosyncracies inherent in the classification systems cited above may explain the discrepancy between the previously (Farkas and Roussos 1984) reported fiber composition of the hamster plantaris muscle (40% FOG and 46% FG) and that reported in the present study (67% IIA, 21% IIX, 0% IIB). 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,

1690 Mattson, Miller, Poole, Delp Figure 1 Percent fiber composition of the costal diaphragm (CD), extensor digitorum longus (EDL), and soleus (S) determined by Farkas and Roussos (1984) according to the methods of Peter et al. (Barnard et al. 1971; Peter et al. 1972; column 1), Wilcox et al. (1989) according to the methods of Brooke and Kaiser (1970; column 2), and by the authors of this paper according to the methods of Schiaffino et al. (1989; column 3). SO, slow oxidative glycolytic; FOG, fast oxidative glycolytic. 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 (Burke 1981; Armstrong and Phelps 1984; Delp and Duan 1996). The general spatial pattern of size distribution was for type I fibers in deep muscles (e.g., vastus intermedius muscle) to be larger than those in more superficial muscles (e.g., vastus medialis muscle). Conversely, type IIX and IIB fibers tended to be largest in superficial muscles. Type IIA fibers did not appear to follow a discernable pattern. In addition, there appeared to be a predictable pattern of fiber distribution within single hindlimb muscles, similar to that in the rat (Delp and Duan 1996). For example, hindlimb muscles with the most heterogeneous fiber composition (e.g., caudal adductor, pectineus, semimembranosus, and gastrocnemius muscles) tended to have a higher concentration of their type I and IIA fibers deep within the muscle, whereas higher concentrations of type IIX and IIB fibers were found in the superficial regions of these muscles (Figure 2). Others have speculated that such spatial variation in both fiber size and type may be related to muscle recruitment activity (Armstrong and Laughlin 1985) and/or economy of energy expenditure (Armstrong and Phelps 1984). 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 Peter et al. (1972), which uses both fiber myosin ATPase activity and metabolic properties to categorize fibers as SO, FOG, or FG, myosin heavy Figure 2 Serial cross-sections of hamster caudal adductor muscle stained red for the presence of type I fibers (top), type IIA fibers (middle), and type IIB fibers (bottom). chain-based fiber typing, as was used in the present study, and myosin ATPase-based typing do not consider fiber metabolic properties as part of the fiber categorization (Reichmann and Pette 1982, 1984; Schi-

Hamster Skeletal Muscle Fiber Composition 1691 affino et al. 1990; Punkt 2002). As a result, there is considerable overlap in the oxidative capacity among fibers typed by their myosin heavy-chain composition or myosin ATPase activity (Reichmann and Pette 1982,1984; Schiaffino et al. 1990; Punkt 2002). 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 (Delp and Pette 1994; Delp and Duan 1996). In addition, the ranges of citrate synthase activity among muscles from the rat and hamster are similar. For example, hamster skeletal muscle citrate synthase activities ranged from 9.9 mol/min/g wet wt in cheek pouch muscle to 64.6 mol/min/g wet wt in the costal portion of the diaphragm (Table 1), whereas in the rat, citrate synthase activity ranges from 8.1 mol/min/g wet wt in cremaster muscle to 42.3 mol/min/g wet wt in the red portion of vastus lateralis muscle (Delp and Duan 1996). In fact, in a muscle in which fiber composition is similar in the hamster and rat (e.g., semitendinosus muscle), muscle citrate synthase activity is almost identical (hamster 13.5 3.8; rat 12.6 0.5 mol/min/g wet wt). In summary, the results of the present investigation demonstrate that the relative 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 (Figure 2). Acknowledgments 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. Literature Cited Aomine M, Arita M, Imanishi S, Kiyosue T (1982) Isotachophoretic analyses of metabolites of cardiac and skeletal muscles in four species. Jpn J Physiol 32:741 760 Armstrong RB, Delp MD, Goljan EF, Laughlin MH (1987) Distribution of blood flow in muscles of miniature swine during exercise. J Appl Physiol 62:1285 1298 Armstrong RB, Laughlin MH (1985) Metabolic indicators of fibre recruitment in mammalian muscles during locomotion. J Exp Biol 115:201 213 Armstrong RB, Phelps RO (1984) Muscle fiber type composition of the rat hindlimb. Am J Anat 171:259 272 Barnard RJ, Edgerton VR, Furukawa T, Peter JB (1971) Histochemical, biochemical, and contractile properties of red, white, and intermediate fibers. Am J Physiol 220:410 414 Brooke MH, Kaiser KK (1970) Three myosin adenosine triphosphatase systems: the nature of their ph lability and sulfhydryl dependence. J Histochem Cytochem 18:670 672 Burke RE (1981) Motor units: anatomy, physiology, and functional organization. In Brooks VB, ed. Handbook of Physiology. The Nervous System. Motor Control. Vol 1. Sec 1. Pt 1. Bethesda, MD, Am Physiol Soc, 345 422 Burns RR, Palade GE (1968) Studies on blood capillaries. I. General organization of blood capillaries in muscle. J Cell Biol 37:244 276 Delp MD, Duan C (1996) Composition and size of type I, IIA, IID/X and IIB fibers and citrate synthase activity of rat muscle. J Appl Physiol 80:261 270 Delp MD, Pette D (1994) Morphological changes during fiber type transitions in low-frequency-stimulated rat fast-twitch muscle. Cell Tissue Res 277: 363 371 Drew JS, Farkas GA, Pearson RD, Rochester DF (1988) Effects of a chronic wasting infection on skeletal muscle size and contractile properties. J Appl Physiol 64:460 465 Farkas GA, Roussos C (1984) Histochemical and biochemical correlates of ventilatory muscle fatigue in emphysematous hamsters. J Clin Invest 74:1214 1220 Faulkner JA, Weiss SW, McGeachie JK (1983) Revascularization of skeletal muscle transplanted into the hamster cheek pouch: intravital and light microscopy. Microvasc Res 26:49 64. Fournier M, Lewis MI (2000) Functional, cellular, and biochemical adaptations to elastase-induced emphysema in hamster medial scalene. J Appl Physiol 88:1327 1337 Gollnick PD, Timson BF, Moore RL, Riedy M (1981) Muscular enlargement and number of fibers in skeletal muscles of rats. J Appl Physiol 50:936 943 Karlinsky JB, Snider GL (1978) Animal models of emphysema. Am Rev Respir Dis 117:1109 1133 Laughlin MH, Armstrong RB (1982) Muscle blood flow distribution patterns as a function of running speed in rats. Am J Physiol 243: H296 306 Laughlin MH, Klabunde RE, Delp MD, Armstrong RB (1989) Effects of dipyridamole on muscle blood flow in exercising miniature swine. Am J Physiol 257: H1507 1515 Lochner A, Brink A, Brink AJ (1971) Protein synthesis in the myocardiopathy and muscular dystrophy of the Syrian hamster. Clin Sci 40:89 94 Lossnitzer K, Kelly TF (1968) Distribution of inulin-carboxyl-c14 in heart and skeletal muscle with respect to in vivo and in vitro extracellular space determinations. Experientia 15;24:126 127 McCurdy MR, Colleran PN, Muller-Delp JM, Delp MD (2000) Effects of fiber composition and hindlimb unloading on the vasodilator properties of skeletal muscle arterioles. J Appl Physiol 89: 398 405 Messina LM (1990) In vivo assessment of acute microvascular injury after reperfusion of ischemic tibialis anterior muscle of the hamster. J Surg Res 48:615 621 Nichols JF, Borer KT (1987) The effects of age on substrate depletion and hormonal responses during submaximal exercise in hamsters. Physiol Behav 41:1 6 Peter JB, Barnard RJ, Edgerton VR, Gillespie CA, Stempel KE (1972) Metabolic profiles of three fiber types of skeletal muscle in guinea pigs and rabbits. Biochemistry 411:2627 2633 Popesko P, Rajtova V, Horak J (1990) A Colour Atlas of Anatomy of Small Laboratory Animals, Vol II. Rat, Mouse, Hamster. London, Wolfe Publishing Punkt K (2002) The difficulties and possibilities of classifying mus-

1692 Mattson, Miller, Poole, Delp cle fiber types in distinct nonoverlapping types. Adv Anat Embryol Cell Biol 162:329 342 Reichmann H, Hoppeler H, Mathieu Costello O, von Bergen F, Pette D (1985) Biochemical and ultrastructural changes of skeletal muscle mitochondria after chronic electrical stimulation in rabbits. Pflugers Arch 404:1 9 Reichmann H, Pette D (1982) A comparative microphotometric study of succinate dehydrogenase activity levels in type I, IIA and IIB fibres of mammalian and human muscles. Histochemistry 74:27 41 Reichmann H, Pette D (1984) Glycerolphosphate oxidase and succinate dehydrogenase activities in IIA and IIB fibres of mouse and rabbit tibialis anterior muscles. Histochemistry 80:429 433 Resch GE, Musacchia XJ (1976) A role for glucose in hypothermic hamsters. Am J Physiol 231:1729 1734 Sarelius IH, Maxwell LC, Gray SD, Duling BR (1983) Capillarity and fiber types in the cremaster muscle of rat and hamster. Am J Physiol 245:H368 374 Schiaffino S, Gorza L, Ausoni S, Bottinelli R, Reggiani C, Larrson L, Gundersen K, et al. (1990) Muscle fiber types expressing different myosin heavy chain isoforms. Their functional properties and adaptive capacity. In Pette D, ed. The Dynamic State of Muscle Fibers. Berlin, New York, Walter de Gruyer, 9 13 Schiaffino S, Gorza L, Sartore S, Saggin L, Ausoni S, Vianello M, Gundersen K, et al. (1989) Three myosin heavy chain isoforms in type 2 skeletal muscle fibres. J Musc Res Cell Motil 10:197 205 Schwartz A, Sordahl A, Crow CA, McCollum WB, Harigaya S, Bajusz E (1972) Several biochemical characteristics of the cardiomyopathic Syrian hamster. Rec Adv Stud Card Struct Metab 1: 235 242 Schwartz LM, McKenzie JE (1990) Adenosine and active hyperemia in soleus and gracilis muscle of cats. Am J Physiol 259:H1295 1304 Sims EA, Landau BR (1967) Diabetes mellitus in the Chinese hamster. I. Metabolic and morphologic studies. Diabetologia 3:115 123 Sodoyez JC, Sodoyez-Goffaux F, Foa PP (1969) Pathogenesis of hypoglycemia in hamsters with a transplantable islet-cell tumor. J Lab Clin Med 73:432 438 Spurway N (1981) Interrelationship between myosin-based and metabolism-based classifications of skeletal muscle fibers. J Histochem Cytochem 29:87 90 Wilcox PG, Hards JM, Bockhold K, Bressler B, Pardy RL (1989) Pathologic changes and contractile properties of the diaphragm in corticosteroid myopathy in hamsters: comparison to peripheral muscle. Am J Respir Cell Mol Biol 1:191 199 Xiao X, Li J, Tsao YP, Dressman D, Hoffman EP, Watchko JF (2000) Full functional rescue of a complete muscle (TA) in dystrophic hamsters by adeno-associated virus vector-directed gene therapy. J Virol 74:1436 1442 Zhan WZ, Sieck GC (1992) Adaptations of diaphragm and medial gastrocnemius muscles to inactivity. J Appl Physiol 72:1445 1453