Uniformity of structural characteristics throughout the length of skeletal muscle fibers.код для вставкиСкачать
Uniformity of Structural Characteristics Throughout the Length of Skeletal Muscle Fibers'.' PATRICIA R. FARRELL AND M. R. FEDDE Neuromuscular Laboratory, Department of Physiological Sciences, Kansas State University, Manhattan, Kansas 66502 ABSTRACT Fresh-frozen, serial cross-sections of the transversus abdominis muscle of four mature chickens (98 fibers) were examined to determine structural and histochemical characteristics throughout the entire length of skeletal muscle fibers. Fiber diameter and nicotinamide adenine dinucleotide diaphorase (NAD-D) and myosin adenosine triphosphatase ( ATPase) activities were used as criteria to classify fibers as Type I or 11. Measurements were made a t 10 to 22 locations along the length of the fibers. An unimodal distribution of mean fiber diameters ranging from 48 I( to 86 p was found. Fibers did not appear larger in the belly of the muscle than near the ends. Although small fluctuations i n fiber diameter occurred through the length of a fiber, large and small fibers tended to remain relatively large or small at each location. NAD-D activity was either consistently high or low throughout the length of a fiber. Likewise, myosin ATPase activity was either high or low for an entire fiber. It is concluded that skeletal muscle fibers maintain rather uniform structural End histochemical characteristics along their entire length. Fibers in a skeletal muscle generally are classified as Type I' or Type 11' depending on histochemical reactions and fiber diameter (Dubowitz and Pearse, '60; Engel, '62, '65; Drews and Engel, '66). Type I fibers have high oxidative enzyme activity, low glycolytic enzyme activity, low myosin adenosine triphosphatase (myosin ATPase) activity, and a smaller diameter than Type I1 fibers, which have low oxidative enzyme activity, high glycolytic enzyme activity, high myosin ATPase activity, and larger diameters. Fibers with enzyme activities and diameters between these two classes have been termed intermediate (Nachmias and Padykula, '58; Beckett, '62; Gauthier and Padykula, '66). The percentages of fibers with various diameters and with certain histochemical characteristics have been determined for several mixed muscles (Nene and Chinoy, '65; Nene and George, '65; George and Berger, '66; Padykula and Gauthier, '67). These values have been based on measurements of cross-sectional samples taken at certain regions within the muscle. However, before measurements of diameter ANAT. REC., 164: 219-230. and cytochemical characteristics of fibers from a single section or region can be used to express the proportion of each fiber type within the muscle as a whole, it is necessary to be certain that these characteristics remain the same within a single fiber regardless of the location along its length at which the section was taken. Our study was designed to determine if muscle fibers in the transversus abdominis of the chicken, many of which course from tendon to tendon, are structurally and cytochemically uniform throughout their length. Characteristics studied were fiber diameter and certain enzyme activities. METHODS Four transversus abdominis muscles (Chamberlain, '43), including the aponeurosis of insertion and bony origin, were removed from four mature, male, White Leghorn chickens killed by an overdose of sodium pentobarbital. The muscles Received Dec. 2, '68. Accepted Jan. 30, '69. 1 Contribution no. 50, Neuromuscular Laboratory, Department of Physiological Sciences, College of Veterinary Medicine KSAES, KSU, Manhattan. Supported by USPHS, NIH grant NB-05786. 219 220 PATRICIA R. FARRELL AND M. R. FEDDE were placed on a saline dampened gauze in a petri dish over ice approximately one hour or until a mechanical stimulus elicited no contraction. A strip of tissue extending from aponeurosis to pubis was excised from the central part of the muscle parallel with the muscle fibers. Tissue strips from birds I and I1 were divided into five pieces of equal length which were positioned adjacent to each other with the ventral transverse planes on corkboard and frozen in 2-methylbutane cooled to - 125" C in liquid nitrogen. Muscle strips from birds I11 and IV were frozen intact, then cut into two pieces, each of which was mounted on a microtome chuck and surrounded by embedding media (O.C.T., - 15 to - 30" C, Ames Lab-Tek, Inc., Westmont, Ill.). All tissue pieces were carefully orientated so cross-sections would be obtained. The blocks were serially sectioned at 10 v with a cryostat at - 15" to - 20" C. Two adjacent sections were picked up on cover slips in each 40 c1 interval (birds I and 11) or 100 c1 interval (birds I11 and IV). One section was stained with hematoxylin-eosin ( H and E ) , while the adjacent section was incubated for histochemical determination of nicotinamide adenine dinucleotide diaphorase (NAD-D) activity in birds I, 11, and I11 (Pearse, '60) or myosin ATPase activity in bird IV (Padykula and Herman, '55). Fibers were much easier to follow when the strips of muscle were frozen prior to blocking and affixing to the cryostat chuck (birds 111, IV), than when the muscle was blocked into pieces and then frozen. Prefreezing resulted in fewer lost sections when blocks were changed and produced less chance for fibers to deviate from true transverse sections. A clearly discernible area of the muscle was traced under a microscope through all the H and E sections. Photographic enlargements of the area were then made at approximately 400 p intervals along the length of the muscle, and individual fibers were identified and numbered on the photographs. Fibers that did not course the entire length of the muscle were not used. Photographs of NAD-D and myosin ATPase sections were also made and cells that had been followed on the H and E photographs were identified. Cross-sectional areas of identified fibers were measured on photographs (370 X enlargement) with a planimeter at ten locations throughout the length of the muscle on 34 fibers in bird I and 27 fibers in bird I1 while those measurements were made at 22 locations on 14 fibers in bird 111; and at 20 locations on 23 fibers in bird IV. The cross-sectional area measurements were then converted to fiber diameter, assuming the fibers to be circular (Gauthier and Padykula, '66). A two-way analysis of variance (Snedecor, '56) was used to determine: (1) if significant differences existed between mean diameters of the various muscle fibers; and ( 2 ) if a significant difference existed between the mean of the diameters at various locations along the muscle. Fibers on photographs of the NAD-D preparations were qualitatively scored as Type I, Type I-intermediate, intermediate, Type 11-intermediate, or Type I1 on the basis of the density of NAD-D localizations. Fibers on the photographs of the myosin ATPase preparations were classified as Type I1 if a predominant dark appearance prevailed or Type I if very little reaction was observed. RESULTS Many fibers of the transversus abdominis muscle in the chicken coursed from tendon to tendon. Figure 1 illustrates that it is comparatively easy to trace an individual fiber over short distances of muscle by observing the size, shape, and relative position within a fasciculus. Sections A and D were only 200 c~ apart. The fasciculi in each photograph are similar and the two fibers marked (0) and (+) maintain approximately the same shape, size, and spatial relation in the muscle. Fig. 1 Spatial arrangement of fibers over a short distance in the transversus abdominis muscle of the chicken. (Bird I. Fresh frozen sections, H and E.) Sections A and B, and C and I 3 are 4 0 p apart: sections B and C are 1 2 0 p apart. Fibers marked ( 0 ) and (+) are the same in each section and were easily identified by similarity in size and spatial relationships. Scale 011 this and following photomicrographs is 35 p . STRUCTURE OF MUSCLE FIBERS ALONG THEIR LENGTH Figure 1 22 1 222 PATRICIA R. FARRELL AND M . R. FEDDE However, neither the configuration of the fasciculi nor the spatial arrangement of the fibers was constant throughout the length of the muscle. Figure 2 shows sections taken 6000 c~ apart. It is not evident from the sections that the labeled fibers are the same in each photograph. That conclusion could only be reached by using close serial sections like those in figure 1. Inspection of the two labeled fibers in figure 2 leads to the conclusion that they retain their relative diameters in each section although the shape and position of the fibers within a fasciculus are not constant over long distances. Relative relationships of three fibers with different diameters are shown in figure 3. Although the diameter of a given fiber fluctuates from place to place, large ones remained large and small ones remained small throughout the length of the muscle. Smallest and largest diameters at a given location of the 98 fibers studied were 30 and 115 u. To determine whether fiber diameters were larger in the belly of the muscle than near the origin or insertion, average diameter of all fibers at each location was computed for birds I11 and IV. Although differences were significant (P < 0.05) in average diameter of fibers at various locations, fibers in the muscle belly were not consistently larger than elsewhere (fig. 4). Differences between mean diameters of fibers in a muscle were very highly significant (P < 0.005). However, there were no distinct populations of small, intermediate or large fibers. A histogram of the mean diameter of 98 fibers from all birds indicates a normal unimodal population of fibers in the transversus abdominis muscle (fig. 5). Sections stained for NAD-D activity (adjacent to the H and E sections presented in fig. 2), are shown in figure 6. Differences between Type I and I1 fibers are less marked than in many mammalian muscles. However, the smaller fibers have more NAD-D activity than the larger ones and other fibers exist between the extreme intensities. The larger fiber ( 0 ) had consistently less oxidative en- zyme activity than the smaller fiber (+) throughout the muscle. Sections illustrating myosin ATPase activity in fibers from bird IV are shown in figure 7. Sections A and D are 16,700 I.I apart. The fiber labeled (+) had low activity at each location; the fiber labeled ( 0 ) had high activity through its entire length. Of the 23 fibers studied in this bird, 18 were Type I1 while five were Type I. The mean diameter of the Type I1 fibers (69.5 rt 2.4 P, S.E.) was significantly different ( P < 0.05) from that of the Type I fibers (62.4 2 2.1 H). DISCUSSION Several lines of evidence indirectly suggest that muscle fibers may contain rePions which differ chemically and morphologically. Segmentation of fibers could occur due to the nature of embryological development since the long multinucleate muscle cell probably forms by successive fusion of individual cells (Konigsberg, '61 ). Furthermore, pathological changes accompanying necrobiotic myopathies include an unexplainable segmented necrosis of some fibers (Adams, '64; Pearson, '65). Also, serial transverse sections of muscle spindles of the rectus femoris of the cat and of the lumbricals of the rat contain structural changes in individual intrafusal fibers (Merrillees, '60; Barker, '62). It has previously been implied that muscle fibers are morphologically, histochemically and physiologically uniform along their length (Nachmias and Padykula, '58; Dubowitz and Pearse, '60, '61, '64; Engel, '65; Drews and Engel, '66; Engel and Irwin, '67). However, the only confirming data which have been presented consist of photomicrographs of longitudinal sections of muscle fibers which were only a few hundred microns Fig. 2 Spatial relationships of fibers over a long distance in tranversus abdominis muscle of the chicken. (Bird I. Fresh frozen sections, H and E.) Sections are 6 0 0 0 ~apart. Fibers marked ( 0 ) and (+) are the same in each section. Although they differ markedly in spatial relationship, they maintain the same relative sizes. STRUCTURE O F MUSCLE FIBERS ALONG THEIR LENGTH Figure 2 223 224 PATRICIA R. FARRELL AND M. R. FEDDE 95 90 a 85 L 80 P) c 75 70 ._ 0 65 60 55 50 1 2 3 4 5 6 7 8 10 9 11 12 13 14 15 16 17 18 19 20 location Fig. 3 Diameters of three fibers a t 20 locations throughout the length of the transversus abdominis muscle. (Bird IV.) Despite fluctuations in fiber diameter each maintains a relative large, intermediate, or small size a t each location. a. 74 72 L 70 E 66 .- 0 66 64 1 ' ~ " " ' ' ' " " " " " ' Fig. 4 Average diameter of 23 fibers at 20 locations throughout the tranversus abdominis muscle of the chicken. (Bird IV.) Despite increased variation of diameters i n the belly of the muscle, no consistent increase in fiber diameter a t any location could be observed. in length. The present study supports these suggestions on the basis of a more thorough examination of the entire fiber length. The transversus abdominis of the chicken is a mixed muscle containing fibers of various diameters and histochemical characteristics. Our data indicate that a given fiber is structurally and cytochemically uniform throughout its length. Fiber diameter remained relatively constant with large fibers tending to remain large and small ones tending to remain small. NADD activity was reasonably constant and myosin-ATPase activity was either high or low for individual fibers. The small fluctuations in the diameter of a fiber along the length of the muscle may have resulted from failure to obtain true cross-sections at each location. An estimate of the reliability of the histological technique used in obtaining true cross-sections was made by plotting deviation percentages in the diameter of a fiber at each location from its mean diameter for fibers of different sizes located close to each other. Similar deviation percentages from the mean for fibers at each location would indicate that the tissue might not be cut in exact crosssection. However, the deviation percentages from the mean diameter were not STRUCTURE OF MUSCLE FIBERS ALONG THEIR LENGTH 225 pected that the diameter of adjacent fibers would alter in unison. Recent investigations regarding the trophic function of nerve on skeletal muscle suggest reasons for the structural and cytochemical uniformity of muscle fibers throughout their length (Guth, '68). There is a rapid increase in ACh sensitivity of the muscle membrane following denervation (Axelsson and Thesleff, '59). Furthermore, innervated end plates in the partially denervated frog sartorius muscle (each fiber of which is dually innervated by two distinct nerve bundles) were found to possess an increased sensitivity to ACh (Miledi, '60; Frank and Inoue, '66) suggesting that the nerve regulates ACh sensitivity throughout the entire length of the muscle fiber. It has also been shown (Guth et al., '66) that reinnervation of a fiber at a distant point from the original sole plate restores the high level of cholinesterase active at that point indicating that the nerve regulates cholinesterase activity throughout the length of the entire muscle fiber. Ions, metabolic energy sources, and proteins all appear to be regulated by specific neural influences (Drahota and Gutmann, '63; Guth and Watson, '67; Mommaerts, '68). Some substance, apparently not acetylcholine, appears to be liberated from the nerve that directs the metabolic and/or contractile properties of the muscle (Miledi, '60, '63; Gutmann, '67, '68). It has 16 been shown that radioisotopically labeled $ C1 4 1 leucine and orotic acid injected into the ventral horn of the spinal cord can travel down the axon of a motoneuron at the rate of 2-3 mm/day and appear in newly synthesized protein and RNA at the nerve ending (Bray and Austin, '68; Peterson, Bray and Austin, '68). If such a substance acts as a controlling factor on the muscle, i t apparently is capable of influencing the entire muscle fiber from its point 45 50 55 60 65 70 75 80 85 90 of release since the present study has Diameter p shown that fibers with very diverse diFig. 5 Histogram of the mean diameter of ameters and cytochemical composition 98 individual fibers from the transversus abdom- maintain relative uniformity throughout inis muscle of four birds. Distinct populations of their length. fibers based on fiber size were not apparent. the same for neighboring fibers of widely varying diameters at many locations in the muscle (fig. 8). In many cases, deviation percentage from the mean diameter of one fiber increased while that of the other decreased. For example, between location 11 and 12 in bird IV, the small fiber (open circles) became smaller while the large fiber (closed circles), only about 70 w away, was becoming larger. It therefore appears that the small variations in diameter at different locations did not result from failure to obtain true crosssection of the fibers. A more plausible explanation for the observed small fluctuations in fiber diameter is that the sarcomere lengths were not constant throughout the length of the muscle. It has been shown that the short sarcomeres, representing some degree of a contraction, are thicker than the longer sarcomeres in the same fiber (Jordan, '33; Brandt et al., '67) and since the muscle was not rigidly fixed during freezing, i t is possible that small degrees of contraction may have occurred at various places along the length of a fiber. If the variations in sarcomere length within a fiber were random, it would not be ex- ::/ IJ 226 PATRICIA R. FARRELL AND M. R. FEDDE Fig. 6 Sections from the transversus abdominis muscle of the chicken illustrating consistency of NAD-D activity throughout the fibers. Sections are 6000 ,u apart; ( 0 ) or ( + ) marked fibers are the same in each section. STRUCTURE OF MUSCLE FIBERS ALONG THEIR LENGTH 227 Fig. 7 Sections from the transversus abdominis muscle of bird IV illustrating myosin ATPase activity in fibers throughout their lenzth. A and B, 6000 $L apart; B and C, 5300 p apart; C and D, 5400 ,u apart. The large fiber ( 0 ) showed positive reaction throughout its length while the showed a comparative negative reaction throughout its length. small fiber (f) 228 PATRICIA R. FARRELL AND M. R. FEDDE 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2 0 location Fig. 8 Deviation percentages of fiber diameters from the mean diameter at the locations studied. (Bird IV.) Two fibers of different sizes (open circles, mean diameter, 56p; closed circles, mean fiber diameter, 84 p ) within approximately 70 p of each other are compared. 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