Morphological histochemical and myosin isoform analysis of the diaphragm of adult horses Equus caballus.код для вставкиСкачать
THE ANATOMICAL RECORD 238:317-325 (1994) Morphological, Histochemical, and Myosin lsoform Analysis of the Diaphragm of Adult Horses, Equus caballus MATTHEW A. COBB, WILLIAM A. SCHUTT, JR,AND JOHN W. HERMANSON Department of Anatomy, College of Veterinary medicine, Cornell University, Zthaca, New York ABSTRACT The horse provides an interesting model for study of the structure and function of the mammalian diaphragm. Multiple regions of diaphragm from seven adult horses were prepared for histochemistry, immunocytochemistry, myosin heavy chain electrophoresis, and native myosin electrophoresis. Two additional adults were dissected to demonstrate myofiber and central tendon morphology and stained for acetylcholinesterase to demonstrate motor endplates. All regions of the adult diaphragm were histochemically characterized by a preponderance of type I fibers with some type IIa fibers. Type IIb fibers were absent in all adult specimens. Myosin heavy chain electrophoresis supported the histochemical study: two isoform bands were present on SDS gels that comigrated at the same rate as rat type I and IIa myosin heavy chain isoforms. No isoform was determined to comigrate with rat type IIb heavy chain isoforms. Native myosin isoform analysis revealed two isoforms that comigrated with rat FM-4 and FM-3 (FM = fast myosin) and two isoforms that comigrated with rat SM-1 and SM-2 (SM= slow myosin) isoforms. In some samples, a third slow native myosin isoform was observed that comigrated at the same rate as the SM-3 of the equine biceps brachii muscle. This doublet (or “triplet”) of slow isoforms is unique to some horse muscles compared with other adult animals studied. It is not known if these multiple slow native myosin isoforms confer some functional advantage to the equine muscles. The adult equine diaphragm also differs in its morphology by having a large central tendon compared to that in other mammals, and is predominantly slow in fiber type and myosin isoform composition. o 1994 Wiley-Liss, Inc. Key words: Horse, Myosin, Muscle, Fiber type As one of the primary respiratory muscles, the diaphragm is required to maintain contractions to facilitate ventilation for the life of the animal. It also must increase the rate and strength of these contractions in response to stress and increased oxygen demand. The diaphragm has been studied intensively, but primarily in such small animals such as rat, cat, and dog (LaFramboise et al., 1991; Gordon et al., 1989; Green et al., 1989; Farkas and Rochester, 1988; LeSouef, et al., 1988; Green et al., 1984; Nystrom, 1968; Gauthier and Padykula, 1966; Sieck e t al., 1983). In light of the fact that horses are a s much as tenfold larger than a dog, and have markedly different abdominal anatomy (in terms of size and position of the abdominal viscera) which may affect diaphragmatic function, the equine diaphragm presents challenges to the traditional concept of diaphragm morphology. Therefore, in this study, we describe the diaphragm in adult horses and test a general hypothesis that the large body size should result in a proportionately large population of slow-twitch and fatigue resistant fibers, thus differing from the diaphragms of small to medium sized mammals. We based this hypothesis on several reports that 0 1994 WILEY-LISS, INC. have noted a trend toward a higher percentage of slow twitch fibers with increasing body size and decreasing respiratory rate (Gauthier and Padykula, 1966; Green et al., 1984), although this relationship is not linear (Farkas and Rochester, 1988). Recently, Rome et al. (the maximum speed of (1990) confirmed that V,, shortening of muscle fibers) of horse soleus muscle fifor muscle fibers from bers was lower than V,, smaller mammals (rats and rabbits). In addition, these in equine soleus fibers authors demonstrated that V,, had a wide range that correlated with differences in myosin heavy chains and light chains associated with three identified fiber types (I, IIa, and IIb). We incorporated several techniques in our study of the adult diaphragm. Gross anatomical studies were used to determine the sampling sites useful in a histochemical study. The results support the hypothesis Received December 29, 1992; accepted September 28, 1993. Address reprint requests to John W. Hermanson, Department of Anatomy, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401. 318 M.A. COBB ET AL. TABLE 1. Adult horses used in this studs Horse 11.2.4 21-4 33.4 43.4 51-3 61-3 71-3 81-3 91-3 Age (yr) 7 6 7 9 6 8 17 7 10 Sex F F F M F F M F M Breed Quarterhorse Standardbred Standardbred Standardbred Thoroughbred Quarterhorse Thoroughbred Standardbred Standardbred Weight (kg) 590 400 500 480 420 560 470 535 490 'Histochemistry. 'Heavy chain electrophoresis. 3Native myosin electrophoresis. 4Hiatal region not sampled. that histochemistry provides a useful indicator of muscle function. However, because the application of standard histochemical techniques to non-traditional species can provide confusing results (Pette and Staron, 1990), our interpretations are supported with electrophoretic separation of myosin heavy chain and native myosin isoforms. Myosin heavy chain isoforms correlate well with functional parameters such as speed of shortening (Reiser et al., 1985). Multiple myosin heavy chain isoforms have been described in the diaphragm of several species such as the laboratory rat (LaFramboise et al., 1991; Termin et al., 1989) and include a fiber type composed of a novel myosin heavy chain isoform now labelled IIx (originally called "IId" by the Termin group). The type IIx myosin heavy chain isoform has been shown to have a n intermediate speed of shortening compared to type I (slow) and type IIb muscle fibers (Schiaffino et al., 1989) and is believed to be associated with repetitively recruited muscles. MATERIALS AND METHODS Morphological studies were performed on fresh muscle specimens obtained from adult horses (Equus caballus) in the postmortem room of the New York State College of Veterinary Medicine (Table 1).Two entire diaphragms were removed from adult horses within 30 minutes of death by barbiturate euthanasia. We used a measuring tape to determine muscle fiber bundle lengths in situ, before dissecting the muscle free. One of these diaphragms was immediately fixed to a rigid surface so that it could be studied further at its in situ length. This specimen was photographed so that surface area measurements could be made. Both specimens were then stained for acetylcholinesterase by the technique of Ypey (1978) to demonstrate the motor endplate distribution patterns within the muscles. Samples for histochemical and electrophoretic analysis were taken from the right costal diaphragm a t the approximate level of the costochondral junction of the fifteenth rib, the right crus of the diaphragm near its origin on the transverse processes of the lumbar vertebrae, and from the left crus of the diaphragm immediately dorsal to the esophageal hiatus. These regions will be referred to as costal, crural, and hiatal, respectively, in this study. Costal and crural samples were obtained from seven animals, and hiatal samples were obtained from five of these animals. Histochemistry was performed following the protocol of Hermanson and Hurley (1990). Samples for histochemistry were taken as 1 to 2 cm3 blocks of tissue and mounted on cork blocks using 5% gum tragacanth. These samples were subsequently frozen in isopentane cooled in liquid nitrogen at -160"C, sealed in plastic bags and stored a t -80°C. Transverse serial sections (10 Fm thickness) were obtained on a cryostat microtome at -20°C and mounted on glass slides for staining. Sections for each muscle were stained for myofibrillar ATPase following preincubation at pH 10.3 for 10 minutes a t 37°C in a NaCl glycine buffer solution or pH ranging from 4.35 to 4.55 in increments of 0.05 for 5 minutes at room temperature in a 0.2M barbital acetate solution. Initial experiments used a wider range of acidic pH from 4.2 to 4.6. Additional sections of each sample were stained with alpha-glycerophosphate dehydrogenase (GPD) to determine glycolytic potential (modified from Wattenberg and Leong, 1960) or with nicotinamide adenine dinucleotide tetrazolium reductase (NADH) to determine oxidative capacity (Novikoff et al., 1961). The GPD samples were incubated in 0.2M phosphate buffer containing 24 mM GPD, 1.2 mM nitro blue tetrazolium, and 2.3 mM menadione for 1hour at 37"C, following the protocol of Hermanson and Hurley (1990) but with the increased GPD concentration and longer incubation. Fibers were classified type I or type IIa according to the method of Brooke and Kaiser (1970). To verify the histochemical results, additional sections of each sample were stained with antibodies to fast and slow myosin. Samples were mounted on glass slides and stored at -20°C. The samples were allowed to warm to room temperature for 10 minutes and then preincubated in a 2% solution of normal goat serum in a 0.1 M phosphate buffered saline (PBS) for 30 minutes in a humid chamber at 4°C. Following preincubation, excess goat serum was shaken off the slide and a n antifast myosin or a n anti-slow myosin primary antibody was applied and allowed to incubate overnight in a humid chamber at 4°C. Fast myosin reacted with Sigma MY-32 (Sigma Chemical, ST. Louis, MO) and slow myosin reacted with S-58 (provided by Dr Frank Stockdale, Stanford University Medical School). The following day, slides were dipped in 0.05 M PBS with 0.085% NaCl and then rinsed in the same solution for 10 minutes. Samples were then reacted with a biotinylated rabbit anti-mouse secondary antibody for 10 minutes, a streptavidin peroxidase enzyme conjugate for 5 minutes, and a n AEC substrate chromagen solution for 15 minutes in the dark. Between each of these steps the samples were rinsed for 10 minutes in the PBS/NaCl solution. After staining, the slides were soaked for 3 minutes in distilled water and coverslipped using a glycerol-vinyl alcohol mounting medium. Black and white photographs (magnifications were 100 x ) obtained using a n Olympus BH-2 photomicroscope were used to determine fiber type percentages and fiber diameters. For each sample, photographs were taken from identical regions of each of the histochemically and immunocytochemically stained samples described earlier for fiber type determination. Seven to ten additional fields of the acid preincubated ATPase stain showing the best contrast between fiber types, typically pH 4.45, were taken for fiber type counts and diameter measurements. For each sample 319 HORSE DIAPHRAGM at least five microscopic fields were counted to provide a minimum of 1,000 fibers to determine fiber type percentages. Thirty-six fibers of each type were measured for least diameter (to the nearest 0.05 mm) using dial calipers. At least five microscopic fields per tissue were sampled in this measurement process. Fiber type diameters and percentages were compared using a n ANOVA followed by a Tukey's comparison test of multiple means (Box et al., 1978). Significance was correlated with P 5 0.05. Myosin heavy chain electrophoresis was performed following the protocol of LaFramboise et al. (1990). Myosin was extracted from minced muscle samples (20100 mg) on ice for 30-40 minutes in four volumes of a high salt buffer (Butler-Browne and Whalen, 1984) (pH 6.5). Extracts were then centrifuged at 13,000 rpm for 30 minutes a t 2°C in a Hill Scientific mv 13 microfuge. The supernatants were then utilized for both native myosin and heavy chain electrophoresis. Supernatant for heavy chain electrophoresis was diluted in 9 volumes of buffer (37% [w/v] EDTA and 0.01% [v/v] 2-mercaptoethanol), vortexed and allowed to precipitate overnight a t 4°C. Centrifugation was repeated and the resulting pellet dissolved in 0.5 M sodium chloride and 10 mM NaPO, and then denatured by immersion in boiling water for 2 minutes. Samples were then diluted 1:lOO in a gel sample buffer (62.5 mM Tris/HCl, 2% [w/v] SDS [sodium dodecyl sulfate], 10% [v/v] glycerol, and 0.001% [w/v] bromophenol blue; pH 6.8). Electrophoresis was performed in a 4.8% separating gel with 30% (v/v) glycerol and a 3% stacking gel with no glycerol. Ten to 15 p,l aliquots of diluted myosin were electrophoresed for 22 hours a t 120 V and 14°C. Separating gels were silver stained according to the protocol of Oakley et al. (1980). The stained gels were photographed on a light table. Native myosin extraction and electrophoresis was performed as described by Butler-Browne and Whalen (1984) and LaFramboise et al. (1990). Native myosin was obtained in the same manner a s for heavy chain electrophoresis. However, following the second centrifugation, a fraction of the supernates was recovered and diluted in 99 volumes of buffer (40 mM sodium pyrophosphate, 50% glycerol, 0.1% [ d v ] 2-mercaptoethanol, pH 8.5 at 4°C). Gels (6 cm length x 0.5cm diameter) were cast in glass tubes 1 day prior to electrophoresis and refrigerated overnight (final gel concentrations 3.88% [w/v] acrylamide, 0.12% [w/v] bisacrylamide, 20 mM sodium pyrophosphate, and 10% [v/v] glycerol; pH 8.6 at 4°C). Electrophoresis was carried out on volumes of extract (10-15 p,l) in running buffer (20 mM sodium pyrophosphate, 2 mM magnesium chloride, 10% [v/vl glycerol, and 0.01% [v/v] 2-mercaptoethanol; pH 8.65 a t 4°C) using a Model 214 Pharmacia vertical gel apparatus. Electrophoresis was performed in a refrigerated room (2"C), at 100 V and 0°C for 18-20 hours. Gels were stained in 0.025% (wiv) Coomassie blue, 7% (v/v) acetic acid, and 2% (v/v) ethanol and destained in 5% (v/v) methanol and 9% (v/v) acetic acid before storage in 7.5% ( v h ) acetic acid. RESULTS Morphological Studies The costal part of the diaphragm attaches to the ribs and costal cartilages on a line passing from the xiphoid process of the sternum, across the costochondral junction of the tenth rib to a point about midshaft on the eighteenth rib. It then turns cranially, reaching the seventeenth rib at the caudal border of its vertebral attachment. There is a thick layer of connective tissue covering the thoracic surface of the entire diaphragm. Virtually all of the muscular part of the costal diaphragm lies directly against the body wall with its fibers running roughly parallel with the craniocaudal axis of the body (Fig. 1). The two crura of the diaphragm attach to the bodies and transverse processes of the first two lumbar vertebrae and are attached to the costal diaphragm by a large central tendon (Fig. 2). The surface area of this central tendon, measured from one preparation, was 34% of the entire surface area of the diaphragm when stretched flat to its in situ length. Acetylcholinesterase staining of two diaphragms revealed 6 to 9 staggered bands of endplates from costal to tendinous insertions in the costal diaphragm. These bands were separated by intervals of about 20 mm. Both crura of the diaphragm showed three bands of endplates evenly spaced from vertebral to tendinous insertions and separated by intervals of about 16 mm. Histochemical Studies The adult diaphragms contained type I and IIa fiber types with type I predominating (Figs. 3,4). Costal and crural areas were similar, containing 66 and 71% type I fibers, respectively (N = 7). The hiatal region contained 84% type I fibers (N = 5), significantly more type I fibers than either costal or crural regions (P < 0.01). The type I fibers were larger than the type IIa fibers in all three regions (P < .05) and size of both fiber types was constant across all three regions sampled (Table 2). Myosin lsoform Electrophoresis All regions of the adult diaphragm demonstrated two isoforms subsequent to heavy chain electrophoresis (Fig. 5) which migrated at the same rate as the type I and IIa isoforms in rat costal diaphragm described by LaFramboise et al. (1991). Native myosin electrophoresis of the adult costal, crural, and hiatal regions showed two or three slow myosin and two or three fast native myosin isoforms (Fig. 6). The slow myosin isoforms resolved correlated with the SM-1, SM-2, and SM-3 isoforms of equine biceps brachii (Hermanson et al., 1991). When only two slow isoforms were present, they were the SM-1 and SM-2 isoforms. The SM-3 isoform was present in one costal, three crural, and three hiatal regions in a total of three horses (animals 6, 8, and 9 in Table 1). The fast isoforms correlated with FM-4 and FM-3 and in six cases FM-2 of the rat diaphragm and horse biceps brachii. The FM-2 isoform was present in 4 costal diaphragms (animals 2, 3, 4, and 5) and three crural samples (animals 2, 3, and 4). DISCUSSION The equine diaphragm demonstrates two interesting morphological characteristics: multiple, irregularly arranged motor endplate bands are particularly evident in the costal region, and a large proportion of surface area is comprised of the central tendon. The endplate banding pattern is similar to the dog and cat as described by Gordon et al. (1989). This pattern suggests 320 M.A. COBB ET AL. Fig. 1. Lateral view of the thoracic and abdominal regions of a horse showing the position of the diaphragm. Craniad is to the left. Muscle fiber orientation is apparent after stripping off a thick sheet of overlying connective tissue (one block was retained to show the differing orientation of the connective tissue fibers relative to the underlying myofibers). The crural portion of the diaphragm is obscured in this view. multiple fibers in series between the costal and tendinous insertions of the muscle. Gordon et al. (1989) described a second endplate banding pattern in the rat and rabbit in which a single continuous endplate band was seen about midway between the costal and tendinous insertions. The horse diaphragm had a higher proportion of its surface area made up of central tendon than the rat, cat, dog, and rabbit described by Gordon et al. (1989). Interestingly, the rabbit most closely resembled the horse in this characteristic (27% of diaphragm surface area in the rabbit compared to 34% in the horse.) The cat, by comparison had a much smaller central tendon (10%). This suggests that size of the central tendon is not simply a function of the size of the animal. This large central tendon and the orientation of the costal diaphragmatic muscle mass along the craniocaudal axis and directly apposed to the body wall suggest that at least this part of the diaphragm acts to pull the central tendinous part directly caudally. This is somewhat different than the classic view of the diaphragm as a dome which is flattened during contraction. In addition, the connective tissue sheet overlying the thoracic surface of the diaphragm is important by imparting significant elastic recoil to the diaphragm (Griffiths et al., 1992). Both fiber type staining and myosin heavy chain electrophoresis of the equine diaphragm revealed a simple two fiber type design which included predominantly type I fibers with fatigue-resistant and oxidative histochemical characteristics. Our histochemical study showed a higher proportion of type I presumed slow fibers than did Zobunzija et al. (1989). However, these authors based their fiber typing on staining intensity for GPD rather than myosin ATPase, and had 5-10% unclassified fibers in each region sampled. In our study, type IIA fibers stained more darkly than type I fibers for GPD, but the resolution was not always good. It was interesting that the horses had no type IIB fibers in the diaphragm despite the presence of these fibers in other equine muscles (Ryan et al., 1992). Other animals which demonstrate type IIB fibers in limb muscles have also demonstrated them in the diaphragm (Reid et al. 1987; Gordon et al., 1989; Green et al., 1984) The type I predominance of the equine dia- HORSE DIAPHRAGM 321 dorsal Muscle shown Motor endplates shown only CVC Caudal vena cava and its tributaries PVC Plica vena cava Sample sites: Costal sample E IJ Crural sample Hiatal sample Fig. 2. Cranial view of the thoracic surface of an adult horse diaphragm positioned flat with muscle fibers at their in situ length. Two insets on the muscle show the motor endplate distributions in costal and crural regions of the diaphragm. Sample sites are highlighted in the costal, crural, and hiatal region (the latter region transmits the esophagus through the diaphragm). 322 M.A. COBB ET AL. Fig. 3.Two histochemical fiber types predominate in the adult diaphragm of the horse. These serial sections were obtained from the costal diaphragm. Fibers were stained for myosin ATPase after preincubation at pH 4.4 (A) or pH 10.3 (B). Note reversal of staining properties for type I and I1 fibers in the two pH conditions. Metabolic properties such as high oxidative potential or glycolytic potential were demonstrated by dark stains following reaction with NADH (C) and GPD (D) protocols. Correlation with the ATPase reactions was provided by antibodies reacting darkly in the presence of slow myosin heavy chains (E) or fast myosins (F).Scale bar equals 100 km. HORSE DIAPHRAGM Fig. 4. Following acidic preincubation (pH 4.4), sections stained for myosin ATPase showed the distribution of type I fibers (darkly stained) for the costal (A), crural (B), and hiatal (C) regions of the adult diaphragm of horses. Scale bar = 100 pm. phragm is consistent with the hypothesized requirements of fatigue resistance and continual activity required for respiration in any mammal and is particularly important for a n endurance athlete such as the horse (Peters, 1989). In addition, the horse has relatively more type I fibers than did the smaller mammals 323 (rat: Green et al., 1989; cats: Sieck et al., 1983; dogs: Reid et al., 1987). The hiatal region contained a significantly higher proportion of type I fibers than either the costal or surrounding crural regions in all adult horses studied. This specialization is consistent with t h a t reported for the cat (Gordon et al., 1989) and dog (Reid e t al., 1987). Reid et al. have proposed that this region may provide a sphincter function for the esophagus. Vomiting is rare in the adult horse, and when seen, is related to very high gastric pressures. Often, vomiting is associated with the terminal stages of gastric dilatation (Smith, 1989). The histochemical features of the hiatal region of the diaphragm suggest i t may contribute to this very strong sphincter function and inhibit emesis. Native myosin electrophoresis of all three regions of the adult diaphragm showed between one and three slow myosin isoforms (SM) which migrated at the same rates as SM-1, SM-2, and SM-3 isoforms of equine biceps brachii muscle (Hermanson e t al., 1991). Identification of this slow triplet isoform pattern has been restricted thus far to the horse where it was observed consistently in the primarily slow lateral head of the biceps brachii but not in the faster medial head. In the equine diaphragm, only the SM-1 isoform was present in all regions of all animals. The seemingly inconsistent expression of the slow triplet isoform pattern raises anew a number of interesting questions concerning the origin, expression, transition, and significance of native myosin isoforms. Hermanson et al. (1991) suggested that the slow myosin triplet represented the retention of fetal or neonatal isoforms in the adult. In consideration of this, a companion manuscript considers the morphology and ontogeny of the diaphragm in foals (Cobb et al., 1994). The question remains, however, why is the slow triplet seen in the diaphragm of some adults, and in some regions of individual diaphragms, while i t does not appear in others? The most plausible explanation is that the SM-2 and especially the SM-3 isoforms are present at low concentrations in the equine diaphragm and are, therefore, a t the limit of resolution for this technique. Another possibility is that retention of neonatal or fetal isoforms is subject to individual variation. A combination of these two explanations may be involved. Bramble and Carrier (1983) showed that respiration and locomotion were directly coupled in a number of species, including the horse. Later cineradiographic and EMG work by Bramble (1989) led to the proposal of the existence of a “visceral piston” effect in which the oscillations of the abdominal viscera, most importantly the liver, caused by the movement of the animal’s body, played a key role in increasing and decreasing the volume of the thoracic cavity. Movement of the liver cranially upon placement of the forefeet causes cranial displacement of the diaphragm and effects expiration. Caudal movement of the liver and diaphragm increase thoracic volume and effect inspiration. In this model of respiration during locomotion, the diaphragm would act to check the forward motion of this piston rather than the classic view of the diaphragm actively contracting to increase the volume of the thoracic cavity. The horse differs in several important ways from the dog which was the animal studied in Bramble’s cineradiographic work. The liver in the horse is a much 324 M.A. COBB ET AL TABLE 2. Fiber types and fiber diameters (pm) in adult horses* Horse No. Costal % type I Dia. type I Dia. type IIa Crural % type I Dia. type I Dia. type IIa Hiatal % type I Dia. type I Dia. tvDe IIa 1 2 5 6 7 8 9 Mean (S.E.) 55 49 46 62 55 49 77 50 43 58 53 46 67 54 43 63 58 49 73 55 46 66 (3) 53 (1) 46 (1) 78 58 47 50 52 43 74 58 46 70 60 50 80 57 50 70 55 39 74 54 48 71 (4) 56 (1) 46 (2) 80 49 41 83 52 46 84 53 43 88 69 47 86 56 48 84 (1) 56 (3) 45 (1) *All type IIa mean diameters were significantly smaller (P 5 0.05) than comparable mean diameters for type I fibers within each of the three regions. Rat DIA (costal) Ha IIX Crural DIA Costal DIA Hiatal DIA - IlbI- Fig. 5. 6% SDS-PAGE of myosin heavy chains (MHCs) from the costal diaphragm of a rat, and the crural, costal and hiatal regions of an adult horse. The four MHCs labelled in the rat sample are shown Adult Rat COS DIA for comparison with the horse samples. Only two MHC isoforms, comigrating with rat type I and IIa MHCs, are present in the adult horse diaphragm. Adult Adult Adult Horse Horse Horse COS DIA CRUR DIA HIATAL DIA SM-1- FM-4FM-3FM-2- FM-4 -FM-3 C- Fig. 6 . A comparison of 4 6 PAGE of native myosin isoforms for the costal diaphragm (COS) of a n adult rat, adult horse (10years old); crural diaphragm (CRUR) and hiatal diaphragm of adult horse (7 and 6 years old, respectively). While rat diaphragm exhibits only one slow native myosin isoform, the adult horse samples exhibit a t least two slow native myosin isoforms. smaller portion of the abdominal mass, and has a relatively smaller area of contact with the diaphragm. The largest part of the abdominal cavity in horses is taken up by the cecum and large (ascending) colon. These structures constitute a massive, freely moveable mass (restrained only by their mesenteric attachments to the dorsal body wall) which is in direct contact with a large portion of the diaphragm. In the dog, the crural diaphragm was the main modulator of the “visceral piston.” The morphology of the horse diaphragm, in which the fibers of the costal diaphragm are arranged so as to pull the abdominal viscera caudally in a “sling” HORSE DIAPHRAGM formed by the diaphragmatic central tendon, places the costal diaphragm in an excellent position to perform that function' In addition, the predominance Of type I fibers in the diaphragm would provide a largely fatigue resistant muscular control over these visceral movements. Arguments against the visceral piston model were recently presented by younget al. (1992), underlining the importance of understanding visceral movements during locomotion and the need for further study. conc~usion,this study provides data highlighting the m o q h o 1 0 0 7 histochemical fiber myosin isoform composition, and presumed fatigue resistance of adult diaphragms. In contrast to other studies of mammalian diaphragms, the horse has a relatively higher proportion of fatigue resistant type I fibers. As such, these data support a n hypothesis that type 1 fibers are prominent in large-bodied mammals. Based on the observations of Rome et al. (1990) that show a tight range of V,, for type 1 fibers in another equine this high number Of I fibers is suited to continuously effect the inspiratory component of ventilation throughout life. Perhaps the coupling of ventilation and ~ocomotion,as suggested by the visceral piston model, reduces the need for including a large Percentage of fast twitch fatigable fibers in the composition of the diaphragm. ACKNOWLEDGMENTS This study was supported by the H.M. Zweig Memorial Fund and benefitted from an NIAID training grant. Drs. Dorothy Ainsworth, William LaFramboise, Ron Meyers, and James Ryan read and commented on an draft Of the manuscript' The manuscript was improved by the comments of two anonymous reviewers. Michael Simmons prepared the figures. Marketa Aschenbrenner provided excellent technical assistance for several phases of the study. The assistance of ~ ~ Daaod and Jacqueline Petrie was invaluable. Ruthie Loeffler patiently worked on word processing of the manuscript. Finally, this work could not have been completed without the encouragement and assistance Of and pathologists at the New State College of Veterinary Medicine. LITERATURE CITED Box, G.E.P., H.Q. Hunter, and J.S. Hunter 1978 Statistics for Experimenters. Wiley, New York. Bramble, D.M. 1989 Axial-appendicular dynamics and the integration of breathing and gait in mammals, zoOl,,29;171-186. Bramble, D.M., and D,R, Carrier 1983 Running and breathing in mammals. Science, 219r251-256. Brooke, M.H., and K.K. Kaiser 1970 Muscle fiber types: H~~ many and what kind? Arch. Neurol., 23r369-379. Butler-Browne, G.S., and R.G. Whalen 1984 Myosin isozyme transitions occurring during the postnatal development of rat soleus muscle. Dev. Biol., 102.324-334. Cobb, M.A., W.A. Schutt, Jr., J.L. Petrie, and J.W. Hermanson 1994 Neonatal development of the diaphragm of horses, Equus caballus. Anat. Rec., 238311-316. Farkas, G.A., and D.F. Rochester 1988 Functional characteristics of canine costal and crural diaphragm. J. Appl. Physiol., 65t22532260. Gauthier, G.F., and H.A. Padykula 1966 Cytological studies of fiber types in skeletal muscle. J . Cell Biol., 28t333-354. Gordon, D.C., C.G.M. Hammond, J.T. Fisher, and F.J.R. Richmond 1989 Muscle fiber architecture, innervation, and histochemistry in the diaphragm of the cat. J. Morphol., 201t131-143. 325 Green, H.J., M.J. Plyley, D.M. Smith, and J.G. Hile 1989 Extreme endurance training and fiber type adaptation in rat diaphragm. J . Appl. Physiol., 66r1914-1920. Green, H.J., H. Riechmann, and D. Pette 1984 Inter and intraspecies comparisons of fiber type distribution and of succinate dehydrogenase activity in type I, IIa, and IIb fibers of mammalian diaphragms. Histochemistry, 81:67-73. Grifiths, R.I., R.E. Shadwick and P.J. Berger 1992 Functional importance of a highly elastic ligament on the mammalian diaphragm. pro,-, R. sot, Lon& [Biol.], 249.199-204. Hermanson, J.W., M.T. Hegemann-Monachelli, M.J. Daood, and W.A. LaFramboise 1991 Correlation of myosin isoforms with anatomical divisions in equine biceps brachii. Acta Anat. (Basel), 141: 369-376. Hermanson, J.W., and K.J. Hurley 1990 Architectural and histochemical analysis of the biceps brachii muscle of the horse. Acta Anat. (Basel), 137t146-156. LaFramboise, W.A., M.J. Daood, R.D. Guthrie, G.S. Butler-Browne, R.G. Whalen, and M. Ontell 1990 Myosin isoforms in neonatal rat extensor digitorum longus, diaphragm, and soleus muscles. Am. J. physiol., 259:L116-L122. LaFramboise, W.A., M.J. Daood, R.D. Guthrie, S. Schiaffno, P. Moretti, B. Brozanski, M.P. Ontell, G.S. Butler-Browne, R.G. Whalen, and M. Ontell 1991 Emergence o f t h e mature myosin Phenotype in the rat diaphragm muscle. Dev. Biol., 144~1-15. LeSouef, P.N., S.J. England, H.A.F. Stogryn, and A.C. Bryan 1988 Comparison of diaphragmatic fatigue in newborn and older rabbits. J. Awl. Physiol., 65:1040-1044. Novikoff, A.B., W. Shin, and J . Drucher 1961 Mitochondria] localization of oxidative enzymes: staining results with two tetrazolium salts. J. Biophys. Biochem. Cytol., 9:47-61. Nystrom, B. 1968 Histochemistry of developing cat muscles. Acta Neurol. Scand. 44t405-439. Oakley, B.R., D.R. Kirsch, and N.R. Morris 1980 A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem., 105:361-363. Peters, S.E. 1989 Structure and function in vertebrate skeletal muscle. Am. ZOO^., 29:221-234. Pette, D., and R.S. Staron 1990 Cellular and molecular diversities of mammalian skeletal muscle fibers. Rev. Physiol. Biochem. Pharmacol., 116t1-76. Reid, M.B., G.C. Ericson, H.A. Feldman, and R.L. Johnson 1987 Fiber types and fiber diameters in canine respiratory muscles. J. Appl. Physiol., 62:1705-1712. Reiser, P.J., R.L. MOSS,G.G. Giulian, and M.L. Greaser 1985 Shortsingle fibers from adult rabbit soleus muscles is lening lvelocity ~ inmyosin correlated with heavy chain composition. J . Biol. Chem., 260;9077-9081, Rome, L.C., A.A. Sosnicki, and D.O. Gable 1990 Maximum velocity of shortening of three fibre types from horse soleus muscle: implications for scaling with body size. J . Physiol. (Lond.), 431 r173185. Ryan, J.M., M.A. Cobb, and J.W. Hermanson 1992 Elbow extensor muscles of the horse: postural and dynamic implications. Acta Anat. (Basel), 144r71-79. Schiaffino, S., L. Gorza, S. Sartore, L. Saggin, S. Ausoni, M. Vianello, K' Gundersen9 and T. 1989 Three myosin heavy forms in type 2 skeletal muscle fibres. J. Muscle Res. Cell Motil., 10r197-205. Sieck, G.C., R.R. ROY,p. Powell, c . Blanco, V.R. Edgerton, and R.M. Harper 1983 Muscle fiber type distribution and architecture of the cat diaphragm. J . Appl. Physiol., 55r1386-1392. Termin, A., R.S. Staron, and D. Pette 1989 Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle. Histochemistryp 92:453-457. Wattenberg, L.W. and JL Leo% 1960 Effects of coenzyme QIO and menadione on succinate dehydrogenase activity as measured by tetrazolium salt reduction. J . Histochem. Cytochem., 8t296-303. Young, 1% R. MCN. Alexander, A.J. Woakes, P.J. Butler, and L. Anderson 1992 The synchronization of ventilation and locomotion in horses (EQUUS caballus). J . Exp. Biol., 166.19-31. Ypey, D.L. 1978 A tpographical study of the distribution of endplates in the cutaneous pectoris, sartoius, and gastrocnemius muscles of the frog. J. Morphol., 155:327-348. Zobundzija, M., Z. Kozaric, Z. Aleckovic, and A. Brkic 1989 Histoenzymatic characteristics of muscle fibers in the horses pars costalis and pars lumbalis diaphragm. Veterinarski Arch., 59t193-202.