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A M . ZOOLOGIST, ll:54S-5">7 (1971). Electron Microscopic Studies of Skeletal and Cardiac Muscle of a Benthic Fish. I. Myofibrillar Structure in Resting and Contracted Muscle LAWRENCE HERMAN Department of Pathology, State University of Neiv York Downstate Medical Center, Brooklyn, New York 11203 AND PAUL DREIZEN Department of Medicine and Program in Biophysics, State University of Neiu York, Downstale Medical Center, Brooklyn, New York 11203 SYNOI'SIS. Electron microscopic studies are reported on glycerinated skeletal and cardiac muscle of a benthic fish, Coryphaenoides species. In white skeletal muscle, the sarcomeres have a resting length of approximately 1.8 p., with thick filaments 1.4 ^ and thin filaments 0.75 fi in length. These dimensions are somewhat shorter than filament lengths of oilier vertebrate muscles, possibly due to the elfect of volume increase during assembly of thick and thin filaments at high hydrostatic pressure. During ATl'-induced contraction of Coryphaenoides muscle from sarcomere lengths of 1.8 ^ to 1.6 jx, there is a characteristic interdigitation of thick and thin filamenLs, with decrease in I band length and no change in length of thick or thin filaments. Howe\er, in sarcomeres contracted to lengths of 1.5 y. to 1.2 p., there is a slight shortening of the A band, apparently due to shortening of thick filaments, that occurs despite the presence of residual ] band in the same sarcomeres. There is no obvious crumpling or distortion of thick filaments during contraction to sarcomere lengths as low as 1.0 /i, but filament organization undergoes extensive disarray at sarcomere lengths approaching 0.7 fi. Although effects from heterogeneity of filament length cannot be excluded with certainty, the present evidence does suggest that contraction ot Coryphaenoideb muscle from 1.6 fi to 1.0 fi sarcomere lengih is accompanied by shoitening of thick filaments consequent to a structural change within the thick filament core. „ ., , , . . ,. Detailed electron microscopic studies .,, , TT i mrr- TT I , n ™ (Hanson and Huxley, 1955: Huxley, 1960, ,V.O. , ,,-,,, , 1963) have established that vertebrate stri,' . . . . . ated muscle contains an interlocking array . .., . , n, , ,? . ; of thick (myosin) filaments and thin (acv ; / i tin) hlaments, with cross-bridges extending & ' . ,. . , , , ,. , „.b at periodic intervals along each thick hla1 5 ... , r „ , ment. It is widely accepted, following the original proposal oi A. F. Huxley and Niedergerke (19o4) and H. E. Huxley and Hanson (1954), that contraction involves a sliding together of thick and thin fila., , . l , ., ments without change in cfilament length, , , .. ° r . , ° and that contractile force is generated by . . . . .. . . .. ' cyclic interaction ol myosin cross-bridges , • • , ,. ,. , and actin sites. According to this theory, , , •• contraction involves a net linear move,• , ., , . . . . . . , . ment ot cross-bridges along individual thin . .,., ° „ ° , , rl hlaments, without structural change in ^ Jeast ^ n d e r l h e ihick fi,amcm h iol icall significant conditions (A. J,_ ; Hux f eyj {95^ H E H u x ] e y > ^ This work was supported in part by research grants from the National Institutes of Health (CA- 19G9). However, some objections have |-, een raised to a simple linear scheme (see 06801 and AM-06165), the Health Research Coun- „ INTRODUCTION - 1 r»'-i\ 1 1 »• 1 1 cil of Xew York Cit>, and the New York Heart Drcizcn, 19/1), and an alternative model Association. P.D. is a Career Scientist of the Health has been proposed, according to which the Research Council of Xew York City. We grate- cros s-bridge interactions result in torsionfully acknowledge the technical assistance of Mr. . . . „. Alan I.ieberman and Miss Catherine Ales. 543 al Winding ofr the thick filament core FIG. 1. Longitudinal si.Ltions of Coryphacnaides white skeletal muscle. (A) Sarcomeres at rest length (1.8 /j), with characteristic Z lines, I bands, A bands, and central M lines. Glycogen bodies are scattered throughout; and remnants of tlie sartoplasmic reticulum (destroyed during glyc- trol extraction) lie between fibril, x 28,400 (B) Sarcomeres o£ different length within the same fiber. Upper region contains sarcomeres of 1.4 ^ length, with narrow I bands; lower region contains sarromeres of 1.1 <r. lenith, with widening of '/. lines. >< 24,000. MYOFIBRILLAR STRUCTURE OF A BENTHIC FISH 545 FIG. 2. Longitudinal sections o£ Coryphaenoid.es white skeletal muscle, showing sarcomeres of length 1.7 n (A) and 1.8 ^ (B). Note the decrease in A band length from 1.38 ^ in the relaxed sarcomere (A) to 1.10 in in the partially contracted sarcomere (B). The I bands persist throughout, so that A band shortening can not be attributed to passive buckling of thick filaments against Z lines. X 70,000. (Dreizen and Gershman, 1970). These general features are derived largely from studies of rabbit and frog skeletal muscles, but similar structural features are found in skeletal and cardiac muscles of other vertebrates (Hoyle, 1969), including those of fish (Franzini-Armstrong and Porter, 1964; Bergmann, 1964; Kilarski, 1967; Nishihara, 1967). This communication describes electron microscopic studies of control and ATP-contracted glycerinated skeletal and cardiac muscle of a benthic fish, Coryphaenoid.es species, whose natural environment includes conditions of temperature close to 0°C and pressure about 3,000 psi. waters near the Galapagos Islands, during the 1970 expedition of the Alpha Helix (see Phleger, 1971). Glycerinated muscle was prepared essentially by the method of Szent-Gyorgyi (1951), as modified by Huxley (1963). Elongated strips of white muscle from the dorsal and lateral trunk, red muscle from the subcutaneous lateral line region, and cardiac muscle (ventricle) were excised, tied to polyethylene rods, and stored in 50% glycerol-0.0067M K2HPO4, pH 7.2, at 4°C. The glycerol solutions were changed at one and two days, and the muscle strips were stored at —20°C for periods of three to four months. Portions of glycerinated muscle were transferred to a solution containing 15% glycerol-85% buffer (0.1M KC1, lmn MgCl2, 0.0067M K2HPO4, pH 7) and allowed to stand for 2-3 hours at 4°C. The muscle was shredded and then homogenized for about MATERIALS AND METHODS CUycerinalcd muscle preparations. The Coryphaenoid.es fish were captured at a depth of 2,200 meters in the off-shore 546 LAWRENCE HERMAN AND PAUL DREIZEN three minutes using a Sorvall Omnimixer at 4°C. Muscle fibers in different stages of contraction were obtained by adding ATP (at 4 mM, 1 HIM, and 0.4 HIM) in 15% glycerol-85% buffer (as above) to an equivalent volume of homogenized muscle fibers in suspension. The fibers were examined with a Reichert polarizing microscope (Xenon source, at 5400 A) for possible changes in striation spacing and pattern of birefringence. Electron microscopy. After incubation in ATP solutions for 30 minutes, the suspension of glycerinated muscle fibers was fixed with 8 volumes of glu tar aldehyde (Polysciences Inc., Rydal, Pa) buffered with 0.1M sodium cacodylate at pH 7.4, for one hour, according to the method of Sabatini et al. (1963). The fixed muscle homogenate was centrifuged at 15,000 rpm for 15 minutes, using a Beckman SW-39L swinging bucket rotor; the pellet was washed three times in 0.2M sucrose-O.lM sodium cacodylate, pH 7.4, for ten minutes. All procedures were done at 4°C, and the samples were stored at 4°C until dehydration in a graded series of alcohols, followed by embedding in Epon (Luft, 1961). Ultra-thin sections were cut on LKB Ultratome I, mounted on uncoated Athene grids, stained with methanolic uranyl acetate (Stempak and Ward, 1964), and examined in a Philips 300 electron microscope. Electron micrographs were taken at 3400, 8700, 27,000, and 65,000 magnifications and photographically enlarged subsequently. RESULTS White skeletal muscle. The general morphology of white skeletal muscle of Coryphaenoides appears similar to comparable muscle of other fish and mammals. Irregularly shaped bundles of myofibrils are separated by clusters of sarcoplasmic reticulum, from which the soluble components were extracted during prolonged storage in glycerol. On longitudinal section, the sarcomere.s show characteristic Z lines, I bands, A hands, and tontral H-zones that are bisected by M lines (Fig. 1A). The resting length of a sarcomere is approximately 1.8 fx,; however, many fibers contain shorter sarcomeres and occasional fibers contain longer sarcomeres. This heterogeneity probably arises from differences in contractile activity prior to death of the fish, but structural variation among the myofibrils cannot be excluded. The sarcomeres are composed of interdigitating thick and thin filaments. At sarcomere lengths close to rest length, the thick filaments are approximately 1.4 JU, in length and the thin filaments are approximately 0.75 /i in length (from center of Z line to free end of the filament). Both sets of filaments are well visualized in crosssections of myofibrils in the region of the A band, where six thin filaments are arranged in an hexagonal array about each thick filament, with an average of two thin filaments per thick filament, as in other vertebrate skeletal muscle (Hanson and Huxley, 1955; Hoyle, 1969). Thick filaments are approximately 110 A and thin filaments are about 40-45 A in diameter. Although cross-bridges may be seen as poorly defined projections of thick filaments in cross section, the cross-bridges are more readily visualized in longitudinal section as periodic densities along the axis of the thick filament or as knobby protuberances that extend towards the thin filaments (Figs. 2, 3). Observations with the polarizing microscope show that addition of ATP results in shortening of glycerinated fibers, with decrease of striation interval from roughly 1.8 /j. to as low as 0.8 ju.. Although the extent of shortening is dependent in general on the concentration of ATP, there is considerable variation in the periodicity of striations within different fiber bundles, ranging from maximal shortening in small fibrils and near the surface of large fibers to little, if any, shortening within the interior of large fibers. The variation in striation pattern presumably arises from a gradient in ATP concentration due to incomplete diffusion of ATP into the muscle fibers under the given experimental conditions. Electron micrographs show compara- MYOFIBRILLAR STRUCTURE OF A BENTHIC FISH 547 3H FIG. 3. Contraction series for Coryphaenoides white skeletal muscle. Sarcomeres are shown at lengths of 1.7 y (A, B), 1.6 y (C), 1.5 y. (D), 1.4 y, (E, F, G), 1.2 y, (H), 1.1 y (I), 1.0 y (J). 0-9 ^ (K), and 0.7 y, (L). These sarcomeres are representative of the large sample of sarcomeres used for the measurements summarized in Fig. 4. Note the slight but progressive decrease in length of A band from 1.38 y, (B and C) to 1.31 y (D, E, F) to 1.26 y, (G), although I band is present in each sarcomere. Thick and thin filaments retain their parallel, ordered array during shortening to 1.0 y. sarcomere length, but prominent contraction bands form and filaments undergo extensive disarray during contraction below 1.0 ix. Approximate location of Z lines are noted in 3L. x 72,000. 548 LAWRENCE HERMAN AND PAUL DREIZEN 31 ble variation in the extent of contraction within different fibrils of the same fiber bundle, as shown, for example, in Fig. IB. The morphological changes which occur during ATP-induced contraction are illustrated in longitudinal sections of white skeletal muscle at sarcomere lengths from 1.7 /* to 0.7 fx. (Figs. 2, .•)). Shortening of sarcomeres from rest length to about 1.4 t, is characterized by interdigitation of thick and thin filaments as Z lines are brought closer together and I bands are progressively shortened (Fig. 3A-G). However, there appears to be slight shortening of the A band in sarcomeres having some residual I band (Figs. 2B, 3D-G). The I band is completely obliterated during contraction to and below sarcomere lengths of 1.2 p (Fig. 311). In sarcomeres contracted to lengths as low as 1.0 //,, there is no appar- MYOFJBRILLAR STRUCTURE OF A BENTHIC FISH 549 extensive disarray of filament organization (Fig. 3L). The effect of ATP-induced contraction •4 fon filament lengths of Goryphaenoidcs white skeletal muscle is summarized quantitatively in Figure 4. The data are plotted without respect to average ATP con•30.4 centration (0.4 HIM, 1.0 mil, and 4 HIM) in the fiber suspension, since a distribution of sarcomere lengths is obtained at each concentration of ATP. During shortening of sarcomeres from 1.8 /*. to 1.6 p, there is linear decrease of I band length, without significant change in lengths of thick or thin filaments, whereas contraction of sar5-1.4 comeres from 1.5 fi to 1.2 p is accompanied by some shortening of the A band. This change is presumably due to shortening of thick filaments, in view of die presence of residual I band and the absence of gross distortion of thick filaments. The I band is 1.0 obliterated during contraction to 1.2 p 1.6 18 1.2 1.4 sarcomere length, and shortening of sarSARCOMERE LENGTH comeres below 1.2 fx, is accompanied by an equivalent decrease in net length of A FIG. 4. Lengths of A band, I band, and thin filaband. Thin filaments do not undergo any ments plotted against sarcomere length for Coryphaenoides white skeletal muscle in the presence significant change in length during conof ATP. Mean values (± standard deviation) are traction of sarcomeres from 1.8 /x to 1.0 ju.. shown. Thin filaments are measured from middle of Z line to free end of thin filament; I Red skeletal muscle. The overall morband length refers to total sarcomere length less phology of red skeletal muscle is similar to A band length, and includes some contribution from Z line protein. that of white skeletal muscle, with myofibrils separated by clusters of sarcoent crumpling or folding of thick filaments plasmic reticulum (Fig. 5). Red skeletal in directions perpendicular to filament muscle contains large, moderately dense axis, although an increased density in the particles that are interspersed between or region of the Z line may possibly represent on myofilaments, without any particular some piling up of the terminal ends of localization within a sarcomere. These parthick filaments (Fig. 3I-J). There is no ticles appear as irregularly shaped bodies evidence for penetration of thick filaments or as solid or doughnut shaped spheroids through the Z line into adjacent sar- and probably represent the glycogen-rich comeres, as described in barnacle muscle stores characteristic of red skeletal muscle, by Hoyle et al. (1965). An interdigitation although the extent and size of these deof thin filaments is evident during contrac- posits greatly exceeds that shown in prevition to 1.0 n sarcomere length, but entire ous studies of fish muscle (Franzinithin filaments are not readily discernible at Armstrong and Porter, 1964; Bergmann, lengths below 1.0 /*. Shortening of sar- 1964; Kilarski, 1967; Nishihara, 1967). Most fibers of red skeletal muscle concomeres to 1.0 ju. and less is accompanied by widening of the Z lines and a partial tain sarcomeres with short I bands or no 1 disruption of the parallel aiTangement of bands at all, although occasional fibers are filaments (Fig. 3K); shortening to sar- seen with sarcomeres as long as 1.9 p.. In comere lengths of 0.7 /x is accompanied by the longer sarcomeres, filament dimensions t-n L2 550 LAWRENCE HERMAN AND PAUL DREIZEN Hi;, b. CoryphaenoUles red skeletal muscle. Filaments contain randomly dispersed, large, round to oval particles of glycogen. Z lines are promi- nent; I bands are lacking; and A bands display poorly defined M lines, x 24,000. are approximately the same as in comparable sarcomeres of white skeletal muscle. Unlike white muscle, red muscle does not contain well-defined M lines, but shows an occasional darkening of the A band mid- way between Z lines. Although red skeletal muscle appears to have undergone extensive contraction during or prior to extraction with glycerol, the fibers undergo further shortening in the MYOFIBRILLAR STRUCTURE OF A BENTHIC FISH FIG. 6. Contracted sarcomeres of Coryphaenoides red skeletal muscle, at lengths of 1.2 p. (A), 1.1 M (B), 0.9 fi (C), 0.9 n (D), 0.7 M (E), and 0.6 M 551 (F). Approximate locations of Z lines are noted in 6E and F. x72,O0O. 552 LAWRENCE HERMAN AND PAUL DREIZEN 70 lit;. 7. Contracted sarcomeres of Goryphaenoid.es cardiac muscle, at lengths of 1.3 p, (A), 1.1 p. (B), 1.0 n (C), and 0.7 p (D). Note occasional penetra- tion of thick filaments through Z line into adjacent sarcomeres in (D); region of Z lines is marked. X 72,000. presence of ATP to sarcomere lengths of Cardiac muscle. The myofibrils of Cory1.1 ix to 0.7 i*. (Fig. 6). As in white skeletal phaenoides cardiac muscle show wellmuscle, there is widening and increased defined Z lines, thick filaments, and thin density of the Z line at sarcomere lengths filaments, but no evidence of M line matebelow 1.1 /j. (Fig. 6A-D), and disarray of rial. Prolonged extraction of cardiac musthe filament structure at sarcomere lengths cle with glycerol results in destruction of most of the mitochondria and sarcoplasmic approaching 0.7 //, (Fig. 6E, F). MYOFIBRILLAR STRUCTURE OF A BENTHIC FISH reticulum; however, their persisting remnants indicate a relatively high ratio of these organelles to myofibrils in Coryphaenoides cardiac muscle. In contrast, in other vertebrate heart muscles, the myofibrils constitute the bulk of the muscle, with minimal amounts of mitochondria and sarcoplasmic reticulum. Glycogen bodies are scattered throughout sarcomeres of Coryphaenoides heart muscle, but to a lesser extent than in red skeletal muscle. As in red skeletal muscle, most sarcomeres of cardiac muscle have already shortened without addition of ATP, although occasional fibers contain sarcomeres with length about 1.8 /x. In the presence of ATP, the sarcomeres of cardiac muscle shorten to lengths below 1.1 ^ (Fig. 7A-D). Contraction to this extent results in the formation of contraction bands which are less prominent than those formed at comparable sarcomere lengths in red or white skeletal muscle. This may be in part related to penetration of some thick filaments through the Z line, as evident in sarcomeres shortened to 0.7 /j. length (Fig. 7D). DISCUSSION Myofilaments of resting muscle. White skeletal muscle of Coryphaenoides contains sarcomeres of rest length ~1.8 ^, with thick filaments of 1.4 ^ length and thin filaments of 0.75 p length. These dimensions are relatively short in comparison with those of the most extensively studied vertebrate muscles, namely, rabbit psoas muscle and frog skeletal muscle, which contain sarcomeres of rest length 2.2 /*, and 2.5 ^, respectively, with thick filaments of 1.6 ix length and thin filaments of 1.0 ju, length (Page and Huxley, 1963). Similar sarcomere dimensions are described in a number of different fish muscles (Kilarski, 1967), although relatively short sarcomeres (~1.6 fj.) have been reported in trunk muscle of black Mollies (FranziniArmstrong and Porter, 1964). These measurements were obtained on muscle prepa- 553 rations fixed with glutaraldehyde, which better preserves true filament dimensions (Page and Huxley, 1963; FranziniArmstrong and Porter, 1964), as compared with earlier studies of osmium-fixed specimens which are characterized by shrinkage of filaments and poorer morphological preservation. Previous studies on frog skeletal muscle show morphological differences between the myofibrils of fast (white) fibers and slow (red) fibers, notably, greater sarcomere lengths in red fibers (although filament lengths are approximately the same in red and white fibers), an absence of well-defined M line in red fibers, and widening of thick filaments at their junction with the Z line (Page, 1965) . Similar morphological differences are found in red and white skeletal fibers of Coryphaenoides, although most of the red skeletal fibers and cardiac fibers appear to have contracted below resting sarcomere length, presumably due to terminal contractile activity of these muscles prior to death of the fish or to subsequent release of products from the extensive sarcoplasmic reticulum of these muscles. It seems unlikely that the relatively short lengths of Coryphaenoides myofilaments represent artifacts due to tissue shrinkage, since specimens of glycerinated rabbit skeletal muscle prepared in this laboratory under identical conditions as Coryphaenoides muscle yielded sarcomeres whose filament dimensions are in keeping with the prior studies of Page and Huxley (1963). Moreover, the intersections of A and I bands and of H and M zones are sharply demarcated in Coryphaenoides muscle, whereas greatly shrunken tissue would be likely to have poorly defined intersections. The well-defined organization of sarcomeres and myofilaments in white skeletal muscle of Coryphaenoides indicates that ascent of the fish from 2,200 meters depth to the ocean surface does not have drastic effect on myofibrillar architecture. There is some evidence that the short filament lengths of Coryphaenoides mus- 554 LAWRENCE HERMAN AND PAUL DREIZEN cles may reflect the high pressures encoun- TABLE 1. Effect of volume change on association of actin and myosin filaments at $,000 psi.* tered under benthic conditions. Previous studies on rabbit skeletal proteins have Myosin Actin shown that the assembly of F-actin from &V 85 cc/mole 300 cc/mole G-actin (Ikkai and Ooi, 1966) and myosin Kp/K° 0.26 0.06 lp o filaments from myosin monomers (AJ -A^ )/monomcr 0.4 kcaJ/mole 1.54 kcal/mole (Josephs and Harrington, 1968) is inhib* Calculations aro based on the equations, ited by high hydrostatic pressure. Thermodynamic analyses indicate a volume AV=-RTl / d In K \) and AF = -RTlnK. change, AF, of 84 cc/mole for actin (Ikkai \ dp JT and Ooi, 1966) and 300 cc/mole for myo- Superscripts p and o refer to pressure of 3,000 psi 1 sin (Josephs and Harrington, 1968) dur- and atmosphere, respectively. ing association into filaments. According to rabbit skeletal muscle. The effect of prespresent concepts of protein structure, a sure might be minimized if the peptide volume increase during association of pro- residues implicated in filament stabilizatein monomers is largely due to the trans- tion were to involve stronger intermolecufer of water from tightly organized clusters lar forces in Coryphaenoides muscle than about hydrophobic or polar sites in mon- in rabbit skeletal muscle, or if the filaomeric protein to more loosely organized ments were to pack more closely with water in free solution as protein sites un- greater overlap per monomer. However, dergo interaction during polymerization preliminary measurements on Cory(Kauzmann, 1959). The effects of salt and phaenoides white skeletal muscle indicate temperature on the assembly of actin and cross-bridge periodicities that are roughly myosin filaments are consistent with a pre- comparable with those found in rabbit dominant involvement of hydrophobic skeletal muscle. groups in actin filaments (Ikkai and Ooi, Structural changes during contraction. 1966) and polar groups in myosin fila- An essential basis for the sliding filament ments (Josephs and Harrington, 1968) . In theory of muscular contraction is the eviboth cases, the effect of a positive A.V dence from electron microscopy of rabbit would be to shift the equilibrium from and frog skeletal muscle that the lengths of filament towards monomer, with increase thick and thin filaments remain in association free energy, AF, of 0.42 unchanged during forced extension of muskcal/mole for actin and 1.54 kcal/mole for cle and during contraction to about 70% myosin during elevation of pressure from of rest length (Hanson and Huxley, 1955; atmospheric pressure to 3,000 psi (Table Page and Huxley, 1963). Yet vertebrate skeletal muscle may shorten from 70% of rest length to about 30% of rest length Comparable volume changes (at least (Huxley and Hanson, 1954; Hanson and qualitatively) might be expected during Huxley, 1955), and contraction to this exfilament formation in Coryphaenoides tent would necessitate concomitant shortmuscle. Thus, under benthic conditions of the A band. In explanation of ening (3,000 psi), the equilibrium would tend to this phenomenon, Huxley (1960, 1965) shift from filament towards monomer, rehas suggested that the terminal ends of sulting in the assembly of thick and thin thick filaments are crumpled as they are filaments with fewer monomers per filadriven against the Z line, although no dement in Coryphaenoides muscles than in tailed evidence seems to have been iThis value is based on a 468.000 molecular presented on this point. During maximal weight for rabbit skeletal myosin (Dreizen et al., contraction of skeletal muscle (30-40% of 1967; Gershman et al., 1969), and is somewhat less rest length), there is disruption of the filathan the AV value of 350-400 cc/mole that was originally estimated by Josephs and Harrington ment lattice and formation of a dense con(1968), based on a molecular weight of 540,000 to traction band in the region of the Z line 600,000 for mvosin. MYOFIBRILLAR STRUCTURE OF A BENTHIC FISH (Hanson and Huxley, 1955; Sjostrand and Jagendorf-Elfvis, 1967). This phenomenon, termed supercontraction, is attained in glycerinated muscle fibers at high ATP concentrations and in fresh muscle fibers following repeated tetanic stimulation (Gordon et ah, 1966) and rapid cooling in the presence of caffeine (Fujii and Sakai, 1969). Supercontraction does not seem to represent a physiological event (Huxley, 1960, 1965; Gordon et ah, 1966). Coryphaenoides skeletal muscle contains sarcomeres with resting length (1.8 n) only slightly greater than A band length (1.4 p), and shortening of A bands occupies a prominent role during ATPinduced contraction. Thus, during contraction of white skeletal fibers (Fig. 4), the length of A band remains constant at sarcomere lengths from 1.8 ix to 1.6 ^, that is, about 85% of rest length. During further contraction from 1.5 jx to 1.2 /* sarcomere length, the A bands undergo slight shortening, despite the persistence of residual I band. This finding is in contrast with earlier studies on rabbit and frog skeletal muscle in which A bands remain constant in length until obliteration of the I band (A. F. Huxlev and Niedergerke, 1954; H. E. Huxley and Hanson, 1954; Hanson and H. E. Huxley, 1955; H. E. Huxley, 1960). The slight shortening of A bands in Coryphaenoides muscle does not seem attributable to shrinkage of thick filaments during fixation, since tissue shrinkage is minimal with glutaraldehyde fixation of muscle (Page and Huxley, 1963; FranziniArmstrong and Porter, 1964) ; nor can the decrease in A band length be attributed to differential shrinkage of thick filaments, since thick filaments of varying length are obtained in the same fibers. Although heterogeneity in thick filament dimensions cannot be excluded on the present evidence, this degree of heterogeneity has not been described in other vertebrate muscles, where filament length appears to be determined within narrow limits by physical-chemical factors (see above) or possibly specific protein components (as yet unspecified). Moreover, thin filament 555 length remains constant in Coryphaenoides fibers, unlike Crustacean fibers, where thick and thin filaments exhibit concomitant differences in length (FranziniArmstrong, 1970). These considerations would suggest that thick filaments undergo slight shortening under conditions in which the Z lines have not yet impinged upon the terminal ends of the thick filaments.2 Furthermore, during contraction of sarcomeres from 1.2 ju. to below 1.0 n in length, the thick filaments extend almost to the middle of the Z line, without obvious folding or crumpling against the Z line, and without other gross structural distortion of the filament lattice or individual thick filaments. These results are difficult to reconcile with any explanation of A band shortening that is based simply on passive buckling of thick filaments against the Z line. Instead, the present evidence on Coryphaenoides muscle would suggest that the thick filament core may undergo some kind of structural change during shortening of sarcomeres below 1.5 ft length. This interpretation does not, of course, question the wellestablished evidence that contractile force is generated by cyclic interactions of myosin cross-bridges with thin filament sites (Hanson and H. E. Huxley, 1955; A. F. Huxley, 1957; H. E. Huxley, 1960, 1969). However, the present interpretation would suggest that cross-bridge interactions have two consequences; a sliding together of thick and thin filaments, and a structural change within the thick filament core which may lead to shortening of the thick filament. If this interpretation be correct, then the conventional sliding filament model requires some modification, at least for Coryphaenoides muscle. In this respect, several 2 In studies on crab muscle, evidence has been reported that ATP-induced contraction of Limulus striated muscle is accompanied by shortening of A bands at sarcomere lengths where I bands have not yet been obliterated (DeVillafranca and Marschhaus, 1963). However, this interpretation has been disputed by Franzini-Armstrong (1970), who has noted the marked variability of filament dimensions in single fibers of the crab Portunus Depurator. 556 LAWRENCE HERMAN AND PAUL DREIZEN recently proposed mechanisms for the contraction of vertebrate skeletal muscle are of interest, as follows: (1) Phase transition, that is, a melting of myofilaments during contraction of muscle, as proposed by Mandelkern et al. (1959). As noted by Hill (1968), however, any such theory would be inconsistent with the unequivocal experimental observation that isometric force is proportional with the number of overlapping sites between myosin cross-bridges and thin filament sites (Gordon et al., 1966). (2) Torsional model. Based on reinterpretation of X-ray diffraction, polarization, and energetic evidence on the contraction of vertebrate striated muscle, Dreizen and Gershman (1970) have proposed the hypothesis that myosin cross-bridges interact with thin filament sites along a helical path, resulting in torsional winding of the thick-filament core within a rigid thinfilament lattice. According to this model, contraction might proceed initially without significant change in thick filament length, but further contraction would be accompanied by shortening of thick filaments. A torsional movement of the thick filament core would be manifest at the molecular level by altering interactions among the myosin molecules and breaking a-helical bonds within the light meromyosin part of individual myosin molecules. 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