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Journal of Phy8iology (1991), 440, pp. 113-129 With 4 figure8 Printed in Great Britain

METABOLIC CAPACITY AND MYOSIN EXPRESSION IN SINGLE MUSCLE FIBRES OF THE GARTER SNAKE

BY R. S. WILKINSON*, P. M. NEMETH, B. W. C. ROSSER AND H. L. SWEENEYt From the *Departments of Cell Biology & Physiology, Neurology and Anatomy & Neurobiology, Washington University School of Medicine, St Louis, MO 63110, and tDepartment of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA (Received 26 November 1990) SUMMARY

1. The transversus abdominis muscle of the garter snake contains fibres of three types: tonic (T), slower twitch (S) and faster twitch (F). Fibre types can be determined by anatomical criteria in living preparations. Individual fibres identified as T, S or F were excised from the muscle and subdivided for two types of biochemical examination. Enzymes of energy metabolism were assayed using quantitative microfluorometric methods. Myosin heavy chain composition was determined by gel electrophoresis. In separate experiments, twitch time-to-peaks of F and S fibres were measured to assess the range of contraction times present within the muscle's twitch fibre population. 2. Metabolic subgroups of fibres were delineated by the relative activities of adenylokinase (AK), lactate dehydrogenase (LDH) and ,-hydroxyacyl-CoA-dehydrogenase (/JOAC). The metabolic subgroups corresponded to the anatomical fibre types. Type F fibres had high levels of enzymes associated with glycolytic (LDH) and high-energy phosphate (AK) metabolism. Type T fibres had high levels of the oxidative enzyme /JOAC. Type S fibres had both types of enzyme activity in intermediate and variable amounts. 3. Three myosin heavy chain isoforms were present in the muscle. Type F and type T fibres each expressed a single isoform, denoted F and T respectively. Type S fibres expressed significant quantities of two isoforms: an isoform unique to this fibre type (denoted S) and the F isoform. 4. Electrophoretic mobility and antibody reactivity of the F myosin heavy chain isoform resembled that of mammalian fast-twitch myosin. By the same criteria, the T isoform resembled mammalian slow-twitch myosin. The S isoform exhibited intermediate characteristics: its antibody reactivity was similar to mammalian fasttwitch myosin, but its electrophoretic mobility was that of mammalian slow-twitch myosin. 5. Based on whole-muscle analysis, two myosin alkali light chains, denoted ALCI and ALC2, and one myosin regulatory light chain were present. Gel patterns suggested that ALCI and ALC2 exist as both homodimers and heterodimers. MS 8961

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6. The population of type S fibres within a given muscle exhibited a much wider range of twitch contraction times than did the population of type F fibres. Diversity of contractile properties among type S fibres may result, in part, from differential coexpression of two myosin heavy chain isoforms, together with highly variable ratios of enzymes from two major metabolic pathways. 7. The clear biochemical distinction among fibre types indicates that each type possesses a unique and limited range of physiological properties. Based on this evidence, it is argued that intrinsic fibre types contribute towards generation of functional diversity within the muscle's fibre population. INTRODUCTION

An important feature of vertebrate skeletal muscle is its ability to operate efficiently over a wide range of speeds and power outputs. This versatility is apparently achieved by a simple mechanism. Each muscle contains a heterogeneous population of muscle fibres organized into motor units having different contractile and metabolic properties; appropriate motor units, each usually containing fibres innervated by one motoneuron, are then recruited for each particular task (Henneman, 1957; reviewed by Stuart & Enoka, 1983, and Zajac, 1990). Two mechanisms are potentially responsible for muscle fibre heterogeneity. The first is differentiation of developing fibres into intrinsic types, with specific innervation by motoneurons of a corresponding type. The second is extrinsic regulation of fibre properties via trophic influence of the innervating motoneuron. In part because the two mechanisms operate concomitantly and are therefore difficult to separate in developmental studies, their relative importance in the establishment of muscle fibre diversity is uncertain (reviewed by Kelly, 1985; Thompson, Condon & Astrow, 1990). An attractive hypothesis is that both mechanisms are significant. For example, a fibre population could be intrinsically divided into a few types, with each type subsequently being subdivided into motor units via innervation by a heterogeneous population of motoneurons. Evidence from adult muscle, in which fibre diversity is already established, has confirmed the importance of neural influence on fibre properties, but the role of intrinsic fibre type is less certain. One muscle exhibits a broad and nearly continuous spectrum of metabolic (Lowry, Kimmey, Felder, Chi, Kaiser, Passonneau, Kirk & Lowry, 1978; Pette & Spamer, 1986) and contractile (Stuart, Binder & Enoka, 1983) fibre properties. Neural influence is at least partly responsible for this diversity, because fibres belonging to the same motor unit share virtually identical properties (Nemeth, Pette & Vrbova', 1981; Nemeth, Solanki, Gordon, Hamm, Reinking & Stuart, 1986; Nemeth, Rosser & Wilkinson, 1991), while those belonging to different motor units in one muscle differ systematically (Nemeth et al. 1991). Similar precise organization at the level of fibre types, as opposed to motor units, has not been demonstrated. Adult muscle fibre populations are usually classified into three to four types, based on any of several metabolic, physiological or anatomical criteria. These include the myosin ATPase histochemical staining reaction (Brooke & Kaiser, 1970), the relative activities of energy-generating enzymes (Lowry et al. 1978), myosin isozyme composition (Staron & Pette, 1987), antibody reactivity of myosin heavy chains (Miller & Stockdale, 1987), physiological properties such as contraction time

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and resistance to fatigue (Burke, 1981) and morphological properties such as endplate size and presence of lipid droplets (Wilkinson & Lichtman, 1985; Wilkinson & Nemeth, 1989). However, the various fibre typing schemes do not always agree. For example, two fibres might be assigned the same type based on myosin ATPase histochemistry, but different types based on energy-generating enzymes (Nemeth & Pette, 1981). This suggests that many more than three to four fibre types exist if fibres are classified according to the combined criteria of two or more typing schemes. Similarly, the presence of at least four myosin heavy chain isoforms in mammalian limb muscles, with various isoform pairs co-expressed in single fibres (Schiaffino, Gorza, Sartore, Saggin, Ausoni, Vianello, Gundersen & L0mo, 1989; Gorza, 1990), indicates that fibre typing based on myosin heavy chains, and by inference, on native myosins or myosin ATPase activity, cannot agree with other schemes in which only three to four types are described (see Staron & Pette, 1986, 1987; Gorza, 1990). These observations allow either of two alternatives. The first is that fibre types divide the spectrum of adult fibre properties into arbitrary categories, which differ according to the classification scheme used. This suggests that fibre types, irrespective of their development role, do not intrinsically divide adult fibre populations into functionally distinct classes, and that neural influence is therefore exclusively responsible for adult motor unit diversity. Alternatively, the properties (and number) of fibre types present in adult muscles may not yet be known, due to limitations in experimental techniques such as the semi-quantitative nature of histochemical or antibody staining, and the relatively small sample size which is feasible when single-cell quantitative biochemical techniques are applied to muscles with large fibre populations. The present study asks whether fibre types contribute towards functional diversity of fibres in the transversus abdominis of the garter snake. The muscle was chosen because its small size and accessibility (- 100 fibres arranged as a single layer) provide several experimental advantages. First, entire fibres can be visualized and excised for precise biochemical analyses. Second, a significant fraction of the fibre population may be studied, such that all putative fibre types present in the muscle are likely to be encountered. Third, the muscle exhibits an alternating pattern of three anatomically distinct fibre classes which is unrelated to patterns of innervation. Existence of this nerve-independent pattern argues that the three fibre classes are due to a developmental programme intrinsic to the muscle (Wilkinson & Lichtman, 1985; Robinson, 1987; see also Miller & Stockdale, 1987). Thus the intrinsic developmental class of each fibre in the adult muscle may be inferred from its anatomical properties. In the present study, individual fibres of the transversus abdominis were characterized in two ways: energy-dependent enzyme content, associated with efficiency of energy utilization, and myosin composition, associated with speed of contraction. Three biochemically unique fibre types were delineated by each method; the types corresponded precisely to each other and to the three intrinsic anatomical fibre classes. The biochemical profiles indicate that fibres of each type have unique, limited and non-overlapping physiological properties. Intrinsic fibre types therefore contribute to functional diversity of fibres within this simple muscle. Part of the work described has appeared in abstract form (Nemeth, Sweeney & Wilkinson, 1988).

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METHODS

Adult garter snakes (Thamnophis sirtalis) were cold-anaesthetized in iced water (10 min) and killed by rapid decapitation. Three contiguous transversus abdominis muscles were dissected from the animal and placed in reptilian saline solution (see Lichtman & Wilkinson, 1987, for details of the dissection procedure). Identification of fibres was performed at room temperature; muscles were then quick-frozen for subsequent biochemical analyses (see below). Elapsed time between killing the animal and freezing was 4-7 h.

Identification of fibres The preparation was placed on the stage of a microscope equipped with Nomarski (differential interference contrast) and epifluorescence optics. The three intrinsic fibre types were distinguished in situ by a combination of anatomical criteria: type of innervation, cellular appearance and size (Wilkinson & Lichtman, 1985). To establish the type of innervation, nerve terminals were stained using the fluorescent probe 4-Di-2-ASP (Magrassi, Purves & Lichtman, 1987) and the main endplate band of the muscle was viewed. Twitch fibres (slower, S; and faster, F) were identified as large fibres innervated by plate-like nerve terminals containing forty to seventy boutons. In contrast, tonic fibres (T) were of smaller diameter and received narrow nerve terminals containing six to thirty boutons. Fibres were then searched along their length for additional endplate sites. Tonic fibres always received several (four to seven) additional terminals, while twitch fibres were innervated at a solitary endplate. Fibres determined to be twitch by the above criteria were next scored as faster or slower twitch. Slower twitch (and tonic) fibres contained refractile lipid droplets giving a 'rough' appearance when viewed with Nomarski optics; the droplets were absent from faster twitch fibres, giving a 'smooth' appearance (Ridge, 1971; Wilkinson & Lichtman, 1985; Wilkinson & Nemeth, 1989). There was considerable variation in lipid droplet density among type S fibres in one muscle, and particularly among muscles from different snakes. About one snake in ten contained S fibres with insufficient lipid for clear identification, particularly after the muscle was lyophilized (see below). These preparations were rejected. After typing a fibre its location was mapped, usually by noting its position relative to either edge of the muscle. Generally all fibres near both edges were typed, and other information, such as fibre diameter, lipid content, and presence of landmarks (e.g. blood vessels), was recorded. This detailed map was sufficient to locate the identified fibres after lyophilization. Once the anatomical type and location of twenty to thirty fibres had been determined, the muscle was pinned to a polystyrene foam block (approximately 2 cm square x 5 mm thick) and submerged in isopentane cooled to the temperature of liquid nitrogen. The frozen muscle was then lyophilized for 24 h at -38°C, at a pressure of less than 10-2 mmHg and stored until use under vacuum at -70 'C. Single-fibre isolation took place in a climate-controlled room (21 TC, 40% relative humidity). Using a stereomicroscope, the previously identified fibres were located and dissected from the muscle sheet using insect pins and razor blade chips. Further details of the freezing and microdissection procedure are given elsewhere (Wilkinson & Nemeth, 1989).

Microfiuorimetric enzyme assays

Isolated fibres were cut into segments 20-50,tm in length for individual enzyme assays. The segments were weighed (10-40 ng weight) on a quartz fibre balance and put into 10,ul of assay reagent suspended in Teflon wells filled with mineral oil. The analytical procedures and assay conditions for measuring the activities of lactate dehydrogenase (LDH), adenylokinase (AK) and ,J-hydroxyacyl-CoA-dehydrogenase (flOAC) are described elsewhere (Hintz, Lowry, Kaiser, McKee & Lowry, 1980). Activities were expressed as mol (kg dry wt)-1 h-' and were the mean of three assays using three 20 ng segments from the same fibre. Coefficients of variation (100 x standard deviation/mean) for triplicate determinations were below 12 % or data were discarded. -

Gel electrophoresis Portions of the same fibres subjected to enzyme assays were analysed for myosin heavy chain isoforms. Segments of 200-500 ng weight were extracted in a sodium dodecyl sulphate (SDS) extraction solution (2-3% SDS, 125 mM-Tris HCl, 30 % glycerol, 5% fl-mercaptoethanol; pH 6-8; 1 4ul per 100 ng of freeze-dried fibre). Samples were boiled at 100 'C for 2 min and loaded onto a

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discontinuous SDS-polyacrylamide gel. The gel system is a variation of that described by Biral, Betto, Danieli-Betto & Salviati (1988), with 40% glycerol in a 6% gel. The gels were run at 50 V for 30 h and then silver-stained using the method of Guilian, Moss & Greaser (1983). Characterizations of native myosin isoforms and of myosin light chain isoforms were performed using whole-muscle extracts. This was necessary because single fibres did not contain sufficient amounts of myosin to be assessed on native gels. To study native myosins the muscle was extracted in pyrophosphate extraction solution (50% v/v glycerol, 1% f,-mercaptoethanol, 5 mM-1,4dithiothreitol, 50 mM-sodium pyrophosphate; pH 89). The tissue was incubated for 15 min at room temperature before overnight storage at 2 0C. Samples were run for 6 h at 120 V in 2-5 mm diameter tubes containing a pyrophosphate polyacrylamide gel using the technique described by Hoh, McGrath & White (1976) as modified by Mabuchi, Szvetko, Pinter & Sreter (1982). To examine the light chain component of native myosins the gels were then split longitudinally. One half was silver stained for native myosin visualization while the myosin-containing segments from the other half were loaded into wells of a 13 % discontinuous SDS-polyacrylamide gel (Laemmli, 1970). The wells were filled with SDS running buffer and the sample allowed to equilibrate for 5 min. The gel was then run at constant current (8 mA) for approximately 2 h and stained with silver.

Myosin purification To confirm the identification of native and denatured myosins, the protein was purified. The entire body musculature of five snakes (primarily intercostal muscles) was dissected and pooled to provide sufficient material. The purification procedure of Persechini & Rowe (1984) was used. This method yields myosin with only trace contamination from other proteins; the contaminants (e.g. actin) are of relatively low molecular weight and therefore easily distinguishable from myosin heavy chains on SDS-glycerol gels. Exactly three myosin heavy chain bands were resolved on such gels. For purposes of comparison crude extracts were also prepared from the pooled whole snake muscle and applied to the native (pyrophosphate) gel system described above. Bands thought likely to contain native myosin were cut from these gels and run on SDS-glycerol gels adjacent to samples of purified myosin. Again, exactly three myosin bands were observed; each band comigrated with one of the bands from the purified sample. Western blots To assess monoclonal antibody reactivity of myosin heavy chains from single fibres, SDS-polyacrylamide slab gels were run, equilibrated in a buffer (12-5 mM-Tris, 96 mM-glycine, pH 6 8) and placed in an electrophoretic transfer device with positively charged nylon. The blotting paper on the side of the gel opposite the nylon was soaked in 1 % SDS to facilitate transfer. Transfer took place at 250 nA constant current, 5 °C, for 20 h. Blots were then incubated with monoclonal antibodies. Immunoreactivity was visualized with a horseradish peroxidase-conjugated rabbitanti-mouse immunoglobulin G secondary antibody reacted with diaminobenzidine. Contraction times The range of twitch contraction times among the twitch fibre population was estimated by measuring twitch time-to-peaks of several fibres per muscle. Fibres were chosen randomly; their types (S or F) were identified as described above. A force transducer (400A, Cambridge Technology, Cambridge, MA, USA) was attached to one tendon (the linea alba) at the centre of the muscle; the other tendon (rib end) was immobilized with magnetic pins. Fibres were stimulated near their endplates (which was found to be the lowest threshold region) via an extracellular glass pipette (tip diameter, 3-6,unm) filled with reptilian saline solution. To elicit a twitch, a single rectangular stimulus pulse (10-30 V, 0-2 ms) was delivered. Ten such twitches were elicited from each fibre studied (0 5 Hz stimulus rate); the last five tension responses were digitized and averaged by a laboratory data acquisition system (TL-1, Axon Instruments, Burlingame, CA, USA). This averaging technique eliminated amplitude variations among the first few twitches from each fibre, and reduced noise. Averaged records were displayed on a video monitor and time-to-peaks measured using movable cursors. Tonic fibres, which respond weakly to single stimuli (Wilkinson & Lichtman, 1985), were not studied.

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A total of forty individual fibres (two snakes) were typed anatomically, excised, and subdivided for biochemical study of enzymes and myosin heavy chains (data summarized in Table 2). Fibres of snake 1 were from three contiguous segmental muscles, while fibres of snake 2 were from a single muscle. Muscles from additional snakes were used to provide extracts for analysis of native myosins, myosin light chain composition, and as a source of myosin for purification. The range of twitch contraction times of S and F fibres was measured in separate experiments using muscles which were not studied biochemically. Anatomical properties Motor endplate morphology unambiguously separated fibres into tonic (T) and twitch groups; the presence or absence of lipid particles in twitch fibres differentiated them into slower (S) and faster (F) twitch types, respectively. The correspondence of these anatomical features with physiological differences among snake fibre types has been shown previously (Ridge, 1971; Wilkinson & Lichtman, 1985). Fibre diameters varied along the length of fibres but were generally largest for F fibres, medium for S fibres, and smallest for T fibres, with some overlap between classes.

Energy-related enzymes To characterize the metabolic capacity of individual fibres, enzymes representing three principal energy-generating systems were assayed: /3OAC (oxidation), LDH (glycolysis) and AK (high-energy phosphate metabolism). Each enzyme was shown in a previous study to exhibit the widest activity range among several tested, and therefore the greatest ability to distinguish individual fibre properties (Wilkinson & Nemeth, 1989). Figure 1 illustrates the relationship among these enzymes (in paired plots) in fibres which were typed anatomically prior to freezing of the muscle. Data from both snakes are shown to indicate the variability which existed among animals in quantitative activity levels of the enzymes. The pattern of activities among fibres in one animal, however, was the same. Fibres fell into three distinct groups (closed polygons in Fig. 1) with either: (1) high /IOAC, low LDH and low AK; (2) high ,#OAC, high LDH and high AK; or (3) high AK, high LDH and low flOAC. The groups corresponded precisely with the anatomically determined fibre types T, S and F, respectively (Table 2). By metabolic classification the three fibre groups of Fig. 1 are oxidative (T fibres), oxidative-glycolytic (S fibres) and glycolytic (F fibres). This partitioning of two principal energy-generating pathways is similar in snake and mammals (Wilkinson & Nemeth, 1989). Intra-type heterogeneity in enzyme activities was largest in the S group of snake 1, with a 3-6-fold range in /3OAC, a 2-1 -fold range in AK and a 1-6-fold range in LDH (Fig. 1A and C). Although less apparent in the S group of snake 2 (Fig. lB and D), we have observed similarly large ranges of activity among S fibres assayed for other studies (Wilkinson & Nemeth, 1989; Nemeth et al. 1991).

Myosin heavy chains In initial experiments, Western blots of SDS-glycerol gels run on whole-muscle extract revealed exactly three myosin heavy chain isoform bands, as did

119 ENZYMES AND MYOSIN IN SNAKE MUSCLE SDS-glycerol gels of purified myosin (see Methods). Fragments of F, T and S fibres were therefore run on SDS-glycerol gels to determine the distribution of the three myosin heavy chain isoforms amongst the fibre types. As shown in Fig. 2, faster twitch fibres contained only the slowest migrating heavy chain isoform, which we

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Fig. 1. Activities (mol (kg dry wt)-1 h-') of three energy-generating enzymes in identified fibres of the tranversus abdominis muscle. Each data point represents one fibre; data from two snakes are shown. Tonic (T), slower twitch (S) and faster twitch (F) fibres fell into subgroups (closed polygons) based on relative activities of LDH (the glycolytic enzyme lactate dehydrogenase; upper plots) or /?OAC (the oxidative enzyme ,-hydroxyacyl-CoAdehydrogenase; lower plots) versus AK (adenylokinase, an enzyme of high-energy phosphate metabolism). Numbered fibres are those whose myosin heavy chain composition is shown in Fig. 2.

refer to as the F isoform. Similarly, tonic fibres contained only the fastest migrating form (the T isoform). Slower twitch fibres in contrast contained two myosin heavy chains, the F isoform, and an isoform which migrated at intermediate velocity, called the S isoform. The S band was usually lighter than the F band and varied in intensity for different S fibres, both among muscles and within the same muscle. In one of fifteen gels run from extracts of slower twitch fibres, the band was absent or undetectable (fibre 20 in Table 2). Because the S band migrated only slightly slower than the T band, and because S fibres comprise only approximately 25% of the muscle (Wilkinson & Lichtman, 1985), the band was not clearly separated in gels run from whole-muscle extract (lanes marked WM in Fig. 2).

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Fig. 2. SDS-glycerol gel (myosin heavy chain region) of identified single fibres from one tranversus abdominis muscle (Table 2, snake 2). Each lane was loaded with extract from one fibre (type indicated below). Extracts of whole transversus abdominis muscle (lanes WM) revealed exactly three heavy chain bands. Faster twitch (F) fibres contained exclusively the slowest migrating myosin heavy chain isoform (uppermost WM band). Tonic (T) fibres contained exclusively the fastest migrating isoform (lowermost WM band). Slower twitch (S) fibres contained two heavy chain isoforms: the isoform found in F fibres, and a third isoform, unique to S fibres, which migrated slightly slower than the T isoform. In whole-muscle extract, the S band was partially obscured by the T band. Fibre numbers correspond to those of Fig. 1 and Table 2.

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Fig. 3. Western blot of SDS-glycerol gel probed with a monoclonal antibody which recognizes all known mammalian and avian myosin heavy chains. Exactly three myosin isoforms were detected in the snake transversus abdominis muscle. Left lane (S + T) was loaded with extracts of one slower twitch plus one tonic fibre, to illustrate separation of the closely migrating S (arrow) and T (lowest band) isoforms. Centre lane (F) was loaded with extract of one faster twitch fibre, which contained only the F isoform (uppermost band). Right lane (WM) was loaded with whole tranversus abdominis muscle extract; the S band (arrow) was partially obscured by the T band.

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Antibody reactivity To investigate the degree of similarity between snake, chicken and rat myosins, Western blots were performed on gels of whole-muscle extract, purified myosin and single-fibre extracts. Myosin-specific monoclonal antibodies which recognize both avian and mammalian myosins were chosen; these were evaluated for possible crossTABLE 1. Immunoreactivity of snake myosins Snake Chicken Rabbit Antibody

F

S

T

Fast Slow Fast Slow twitch twitch twitch twitch + + + + + + + +

PAN + + + P08 + + + + + + Plo + + + + F28 + + + + F57 + + + S1o + + + Reactivity of monoclonal antibodies with reptilian (garter snake) myosin heavy chains. Data are from Western blot analysis of SDS-glycerol gels. Only antibodies which recognized both mammalian (rabbit) and avian (chicken) myosins were tested. PAN (Amersham 1169; see Fig. 3), P08 and PIO are pan myosin antibodies which recognize all known mammalian and avian myosin heavy chain isoforms (H. L. Sweeney, unpublished). These antibodies recognized all three myosin heavy chains found in snake muscle. However only three of fifteen fast twitch (F28 and F57) or slow twitch (S1O) specific antibodies recognized snake myosin. Snake F myosin was antigenically similar to mammalian and avian fast twitch myosin, while snake T myosin was antigenically similar to mammalian and avian slow twitch myosin. Snake S myosin reacted only with antibody F28, suggesting that it more nearly resembles fast twitch myosin than slow twitch myosin of other species. Antibodies were mouse anti-rabbit (P08, F57, S1O) or mouse anti-chicken (PIO, F28). -

-

-

reactivity with snake myosin. The majority of the antibodies tested failed to recognize snake myosin. However, three monoclonal antibodies which recognize all known sarcomeric myosins in rat and chicken (H. L. Sweeney, unpublished) recognized exactly three bands, those designated above as F, S and T. One of these antibodies (Pan myosin antibody RPN 1169, Amersham Corp., Arlington Heights, IL, USA) is known to react with myosin I and myosin II classes of heavy chains from both plants and animals (Parke, Miller & Anderton, 1986). A blot utilizing this antibody is shown in Fig. 3. As in the silver-stained gel of Fig. 2, the three isoforms were not well-separated in lanes run from whole-muscle extract. One lane in the gel was therefore loaded with a mixture of extracts from a tonic fibre (containing the T isoform) and a slower twitch fibre (F and S isoforms) to show separation of the closely migrating S and T heavy chain bands. As summarized in Table 1, the use of various monoclonal antibodies to probe Western blots suggested that the fastest-migrating (T) myosin heavy chain isoform is antigenically related to the mammalian and avian slow-twitch myosin heavy chain class. In contrast, the slower migrating forms (F and S) were recognized by fast-twitch specific mammalian and avian antibodies.

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Native myosins and myosin light chains As discussed in the Methods, single fibres did not contain sufficient myosin for characterization of light chains or of native myosin with our gel systems. However, some information was obtained using myosin extracted from whole muscles.

* 290 24.0

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Light chains Fig. 4. Two-dimensional gel analysis of myosin light chain composition in whole transversus abdominis muscle extract. Native gel (left) revealed three diffuse native myosin bands (A, B, C). The bands were cut from the native gel and run on an SDS-glycerol gel to resolve light chains (lanes A, B, C at right). Each native band was found to contain the same regulatory light chain (lower bands, 19 kDa; molecular weight standard MW at centre). The slowest migrating native band (A) contained a single 26 kDa alkali light chain (ALCl, lane A). Similarly, the fastest migrating band (C) contained a single 24 kDa alkali light chain (ALC2, lane C; slightly contaminated by native band B; see text). The third native band (B) migrated slightly slower than native band C. This band contained both light chains (lane B) in approximately equal amounts. Native

-

-

Purification of snake native myosin, followed by SDS gel electrophoresis, revealed the three heavy chain bands described above, and, in addition, three light chain bands of approximately 19, 24 and 26 kDa (see below). Based upon its molecular weight and the fact that it can be phosphorylated (H. L. Sweeney, work in progress), the 19 kDa light chain is of the class referred to as regulatory (Kendrick-Jones, Szentkiralyi & Szent-Gyorgyi, 1976). The two higher molecular weight light chains are assumed to be the alkali light chains. We refer to the 26 kDa light chain as ALCI and the 24 kDa light chain as ALC2. Whole-muscle extracts were also used for study of native myosin. Pyrophosphate gels resolved three diffuse native myosin bands (Fig. 4, left panel). Each of the bands was cut from the native gel and subjected to SDS gel electrophoresis to determine its heavy and light chain content. All three heavy chains appeared to be present in each of the native band, although it was not possible to discern the S isoform band from the more predominant T isoform band with certainty (data not shown). In contrast, -

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the native myosin bands differed in light chain composition, which evidentally determined, at least in part, their differential mobility (Fig. 4, right). The slowest migrating band on native gels contained only ALCL (ALCl homodimers, lane A). The intermediate band contained equal amounts of ALCI and ALC2 (ALC1:ALC2 heterodimers, lane B). The fastest migrating native band contained only ALC2 TABLE 2. Summary of fibre properties Snake Fibre no. Anatomical type Metabolic type Myosin isoforms 4 T Ox T T Ox T 7 T Ox T 10 T Ox T 13 Ox T T 15 Ox T T 18 2 Ox, Gly S, F S 3 S Ox, Gly S, F 8 S Ox, Gly S, F 9 S Ox, Gly S, F 12 Ox, Gly S, F S 14 S Ox, Gly S, F 17 S Ox,Gly S,F F F 5 Gly F F 6 Gly F 11 F Gly F F 16 Gly F F 19 Gly Ox 2 2 T T Ox T T 4 Ox T T 7 Ox T T 9 Ox 11 T T Ox T T 13 Ox T T 15 Ox 21 T T 1 Ox, Gly S, F S 3 S Ox, Gly S, F 6 S Ox,Gly S,F 10 S Ox, Gly S, F 14 S Ox, Gly S, F 18 Ox,Gly S,F S F 20 S Ox, Gly F F 5 Gly F F 8 Gly F F 12 Gly F F 16 Gly F F 17 Gly F F 19 Gly 22 F F Gly Anatomical types are: T, tonic; S, slower twitch; F, faster twitch. Metabolic types are: Ox, oxidative; Gly, glycolytic; Ox, Gly, oxidative-glycolytic. Criteria for anatomical and metabolic fibre typing are described in the text. Fibre 1 of snake 1 was lost while being weighed.

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(ALC2 homodimers, lane C). The trace amount of ALCI in this band is presumed to be contamination from incomplete exclusion of the adjacent heterodimer band when the native gel was cut. Similar contamination, but to differing degrees, was seen in each of four experiments in which separation of the diffuse native bands was attempted. Twitch contraction times Because myosin is one of the proteins which determine twitch contraction time, it was of interest to determine if S fibres, which expressed two myosin heavy chain isoforms, exhibited a wider range of contraction times than did F fibres. The range of contraction times in a particular muscle was therefore estimated from measurements of twitch time-to-peaks for ten randomly chosen fibres of each type (about one-third of the twitch fibre population) in each of three muscles (A-C). Muscle A was from the same snake (snake 1) as muscles studied biochemically; muscles B and C were from snakes not studied biochemically. In muscle A, the range of contraction times for S fibres was 50-75 ms (range, 25 ms), while that for F fibres was 43-52 ms (range, 9 ms). In muscle B, S fibre contraction times ranged from 52 to 80 ms (range, 28 ms) while F fibre contraction times ranged from 40 to 46 ms (range, 6 ms). In muscle C, S fibre contraction time ranged from 60 to 90 ms (range, 30 ms), while F fibre contraction times ranged from 44 to 55 ms (range, 9 ms). Thus the range of S fibre contraction times (average, 28 + 3 ms; mean + S.D.) was significantly larger than the range of F fibre contraction times (average, 8+ 2 ms). DISCUSSION

Individual snake muscle fibres were characterized by three independent means: anatomical appearance in the living state, metabolic enzyme profile and myosin heavy chain composition. All fibres typed anatomically as F, S or T were assigned to a corresponding class by metabolic criteria. Similarly, with one exception (fibre 20 of snake 2), each fibre was assigned to a third corresponding class on the basis of myosin heavy chain content. A large fraction (about one-fifth) of the fibre population was studied, making it unlikely that fibres having properties other than those described were present in the experimental muscles. Exactly three fibre types were therefore shown to exist. Each type exhibited a unique pattern of metabolic and contractile protein expression, and, by inference, a unique range of functional capacity. Metabolic enzymes Assays of enzyme activities provided quantitative information about metabolic properties of individual fibres. The assays not only established three metabolic fibre types, but, in conjunction with gel electrophoresis, permitted direct comparison of metabolic and contractile proteins at the cellular level. Enzyme profiles of fibres did not segregate into non-overlapping groups that would allow unambiguous fibre type identification on the basis of one enzyme alone. However, by utilizing pairs of enzymes with wide activity ranges (LDH or /?OAC versus AK), fibre types were wellseparated in two-dimensional plots. Each metabolic group exhibited a unique enzyme profile with regard to its mean enzyme activities, and, more significantly,

ENZYMES AND MYOSIN IN SNAKE MUSCLE 125 with regard to the type of variability seen among fibres within the group. T fibres contained little AK and LDH; the variability of these slow, oxidative fibres was primarily in the activity of /JOAC. Similarly, F fibres contained little flOAC, but expressed large and variable amounts of AK and LDH. In contrast, S fibres contained all three enzymes in intermediate and variable quantities. Thus differences in enzyme activities reflected differences in absolute metabolic capacity among fibres of all three types, but only S fibres exhibited a wide range of relative capacities for oxidative versus glycolytic (and high-energy phosphate) metabolism.

Myosin composition The difference in myosin composition among the three fibre types was clear. Type F and T fibres each expressed a single detectable heavy chain isoform (F and T, respectively), while each type S fibre expressed two isoforms (F and S) in measurable amounts. Thus fibres could be classified into exactly three types on the basis of apparent on-off 'switches' for myosin heavy chains. The existence of three discrete myosin heavy chain classes, and the correspondence of each to a fibre type assigned independently by both anatomical and metabolic criteria, argues that each fibre type represents a distinct phenotype, as opposed to artificial subdivision of a continuum of fibre properties into three categories. These phenotypes could result from the neurogenic influence of three putative classes of motoneurons (either transiently during development, or continuously throughout adult life). Alternatively, the phenotypes might reflect different lineage histories, independent of innervation (see Miller & Stockdale, 1987). This possibility is consistent with a previous conclusion, based on the alternating fibre type pattern present in the transversus abdominis muscle, that snake fibre types differ intrinsically (Wilkinson & Lichtman, 1985; Lichtman & Wilkinson, 1987; see also Robinson, 1987). Qualitatively, the pattern of myosin expression among the three fibre types paralleled that of metabolic enzymes. F and T fibre types each expressed a single detectable myosin heavy chain isoform, and exhibited capacity for a single predominant type of metabolism (oxidative versus glycolytic). In contrast, S fibres contained two heavy chain isoforms, together with highly variable capacity for both oxidative and glycolytic metabolism. Variability in band density (particularly the S isoform band) suggested that the myosin isoform composition of S fibres was also variable, although this point remains speculative without proper quantification. Presumably, the two myosins differ in contractile properties. In rabbit soleus muscle, for example, some fibres contain both slow (type I) and fast (type IIA) myosin heavy chains (Staron & Pette, 1986); the maximum shortening velocity of these fibres depends on the relative concentration of the two isoforms (Rieser, Moss, Giulian & Greaser, 1985). Muscle of frog (Edman, Reggiani, Schiaffino & te Kronnie, 1988), chicken (Miller & Stockdale, 1987; Rieser, Greaser & Moss, 1988) and humans (Biral et al. 1988) have also been shown to contain at least some fibres in which two myosin heavy chain isoforms are present. However, it is not year clear if such fibres comprise a significant portion of the adult fibre population in those animals. In contrast, the reptilian S fibres are a predominant (25 % of fibres in the transversus abdominis muscle; Wilkinson & Lichtman, 1985; see also Ridge, 1971) adult fibre type in which two myosin heavy chains are consistently co-expressed.

R. S. WILKINSON AND OTHERS 126 The three myosin heavy chains in snake muscle failed to cross-react with the majority of antibodies tested (all of which recognized both avian and mammalian myosin). However, the subset of antibodies which did cross-react gave a consistent picture. The snake type F myosin heavy chain is related to the fast-twitch class of myosin heavy chains found in avian and mammalian muscle, based on both antibody reactivity and on electrophoretic mobility on SDS-glycerol gels. By the same criteria, the T heavy chain is related to the mammalian and avian slow-twitch class. The class of the third snake heavy chain, which is unique to S fibres, is less clear. Based on the antibodies assessed to date, the antigenic similarity is that of the fasttwitch class. However, the electrophoretic mobility is that of the slow-twitch class. Thus S myosin appears immediate between mammalian slow and fast heavy chain classes. As such it is likely to possess intermediate contractile properties compared to snake T and F myosins, consistent with the intermediate contraction times exhibited by S fibres (Wilkinson & Lichtman, 1985). Differences between snake and mammalian myosins were also evident in the native molecule. The three bands on pyrophosphate gels prepared from snake muscle extracts were diffuse compared to bands on gels prepared from mammalian muscle. One explanation is that mobility of the snake native myosins was primarily affected by the light chains but influenced by the heavy chains as well. Analyses of myosin light chains associated with each native myosin were consistent with this interpretation: each of two native bands contained one of the alkali light chains present in purified snake myosin, while a third band, which migrated at intermediate velocity, contained both light chains. When this band was loaded onto a denaturing gel, the two light chain bands appeared at approximately equal intensity, suggesting that they comprise a heterodimer associated with one or more of the heavy chains. Because our native gel system was not sufficiently sensitive to assay single cells (with known heavy chain content), the associations between the three possible light chain combinations (two homodimers and a heterodimer) and the three fibre types in the muscle are not yet known. Relation of enzyme and myosin expression to motor units Fibre types in the transversus abdominis muscle differ systematically in their patterns of innervation. The muscle's approximately twenty-five type S fibres comprise four to five motor units which vary systematically in size, similar to the motor unit arrangement of mammalian muscle (Lichtman & Wilkinson, 1987; see also Hammond & Ridge, 1978). Enzyme content and twitch time-to-peaks are similar among fibres belonging to one motor unit, and differ systematically among different size motor units in one muscle (Nemeth et al. 1991). Thus, although the intrinsic functional capacity of S fibres is limited by the range of metabolic and contractile proteins expressed, properties of a particular fibre within this range are determined by the innervating motoneuron. In contrast, the approximately twentyfive type F fibres form either a single large motor unit or two units with similar contraction times (Lichtman & Wilkinson, 1987; R. S. Wilkinson, unpublished). Lastly, each of approximately fifty type T fibres is probably innervated, with approximately equal strength, by all three tonic motor axons present in the muscle (Lichtman & Wilkinson, 1987). Tonic fibres therefore comprise what is in effect a single motor unit commanded by the integrated activity of three motoneurons. Thus

ENZYMES AND MYOSIN IN SNAKE MUSCLE 127 only the S fibre population is subdivided into motor units of systematically differing properties. Interestingly, only S fibres contained two myosin heavy chains, providing them with a simple mechanism for neural control of contractile properties. The fact that type S fibres exhibited a 3-fold greater range of contraction times than did type F fibres suggests that this mechanism is utilized. Neural control might be achieved by regulation of one heavy chain isoform, or differential regulation of both isoforms, via activity patterns or other trophic influence transmitted at the nerve-muscle synapse. Further study of these possibilities will require precise measurement of contractile properties (e.g. shortening velocity) together with quantitative assays for S and F heavy chain isoform content in single type S fibres. The present results, taken together with a previous motor unit study (Nemeth et al. 1991), indicate that the fibre diversity required in this small reptilian muscle results from a combination of two mechanisms: first, the primary division of the muscle into three intrinsic fibre types, and second, the subdivision by neural influence of one fibre type (slower twitch) into motor units with differing properties. One interpretation of these results is that fibre types play a similar role in larger mammalian muscles, excepting that fibres of all types are subdivided to achieve the required number of heterogeneous motor units. We thank T. Jordan for excellent technical assistance and J. Lichtman for critical comments. This work was supported by National Institutes of Health grants NS-24752, DK-38375 and AR35661 and by the Muscular Dystrophy Association. REFERENCES

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