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The Myofilament Lattice : Studies on Isolated Fibers

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The Myofilament Lattice : Studies on Isolated Fibers THE MYOFILAMENT LATTICE : STUDIES ON ISOLATED FIBERS I. The Constancy of the Unit-Cell Volume with Variation in Sarcomere Length in a Lattice in which the Thin-to-Thick Myofilament Ratio is 6 :1 ERNEST W . APRIL, PHILIP W . BRANDT, and GERALD F . ELLIOTT From the Department of Anatomy, College of Physicians and Surgeons of Columbia University, New York 1003, Department of Chemistry, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213, and the Marine Biological Laboratory, Woods Hole, Massachusetts 02543 . Dr. Elliott's pres- ent address is The Open University, Walton Hall, Bletchley, Buckinghamshire, England ABSTRACT The spacing between the thick myofilaments of muscle fibers from the walking legs of cray- fish (Orconectes) was determined by optical transform analysis of electron micrograph plates of fixed single fibers and by X-ray diffraction of living single fibers . Sarcomere lengths were determined by light diffraction prior to fixation and prior to the in vivo experiments . From these combined measurements, it is demonstrated that the unit-cell volume of the myo- filament lattice is constant during muscle shortening, indicating that the myofilament lattice works in a constant-volume manner. It is further demonstrated with X-ray diffraction measurements of living single fibers that the myofilament lattice continues to work at constant volume after the sarcolemma is removed from the fiber . This indicates that the con- stant-volume behavior of muscle is inherent to the myofilament lattice . INTRODUCTION There is general agreement that the structural root of the sarcomere length . These results were basis for striated muscle shortening involves an confirmed and extended to include active muscle interdigitation of the thick and thin myofilaments by Elliott et al . (8, 10-12) . Carlson et al . (5) as first proposed by A . Huxley and Niedergerke observed that the myofibrils of fibers studied by (20) and H . E . Huxley and Hanson (26) . Con- electron microscopy are larger in cross-section at comitantly, there is a change in the distance shorter sarcomere lengths, but not according to separating the myofilaments . H . E. Huxley (22, a strict constant-volume manner for they reported 24) showed from X-ray diffraction diagrams that a fluid loss of up to 20 %. More recently, Brandt the in vivo lattice spacing of relaxed vertebrate et al . (3) found that the lattice volume is constant muscle is inversely proportional to the square in frog semitendinosus single fibers fixed at a 72 THE JOURNAL OF CELL BIOLOGY • VOLUME 51, 1971 . pages 72-82 variety of sarcomere lengths for electron micro- Measurement of Sarcomere Length by scopic study . Light Diffraction This report demonstrates with data obtained by electron microscopy and low-angle X-ray The diffraction of light by the regular sarcomere diffraction that a constant-lattice-volume regimen repeat of muscle was utilized to determine accurately exists in crayfish muscle fibers which have a the sarcomere length (see, for example, 35) . A 0 .3- lattice configuration markedly different from that mw He-Ne laser (wavelength 6328 A ; Optics Tech- nology Inc., Los Angeles, Calif., Model 170) was of vertebrate muscle (April and Brandt, manu- mounted on an optical bench with a leaf shutter, the script in preparation) . This constant-volume muscle fiber, and a Polaroid packet holder which was (isovolumic) behavior is maintained after the equi- approximately 7 cm beyond the fiber . The exact librium volume is changed osmotically and even specimen-to-film distance was determined from an after the sarcolemma is removed from the fiber . optical transmission grating placed in the saline This precise geometrical variation in lattice solution in the specimen plane . The muscle chamber spacing with sarcomere length is of interest because and hence the fiber could be positioned in the laser the underlying mechanism is not understood beam by rack and pinion accessories . The beam in- and the relation between the interfilament distance tensity was modulated with neutral density filters so and force development in muscular contraction has that the diffraction pattern could be recorded in %0th-%5th sec on Polaroid type 57 film yet to be determined . Brief accounts of these . The muscle fiber could be exposed, however, to the unfiltered results have already appeared (1, 2, 2 a) . beam for several hours without apparent injury . The diffraction patterns (Fig . 1) were measured MATERIALS AND METHODS on the Polaroid print with a 7 X magnifying microm- eter. Because the sarcomere length in these muscle Dissection of Single Muscle Fibers fibers is of the order of 10 µ, the angle of diffraction Single fibers were dissected from the carpopodite is very small (about 4 °) and the sarcomere length (L8 ) flexor which lies in the meropodite of the walking leg is inversely related to the measured distance between the selected order of diffraction and the of crayfish (Orconectes) . The dissections were ac- complished according to the method described by incident spot according to the grating equation : Girardier et al. (18) while the muscles were bathed n X = d in a crayfish physiological saline solution in a plexi- sin B, approximated as n X = L8 tan 0 glass chamber fitted with a glass back . The physio- The strain transducer (Grass Instrument Co., logical saline solution was a modification of the van Quincy, Mass ., Model FT . 03C) was mounted with a Harreveld crustacean saline solution (36), containing micrometer mechanism to the platform carrying the 200 mm/liter NaCl, 13 .5 mm/liter CaCI 2 , 5 mm/liter muscle chamber . The muscle fiber length and hence KCI, and 1 mm/liter NaHCO3 , or 2 mm/liter Tris . the sarcomere length was adjusted by moving the The proximal end of the muscle fiber was left at- strain transducer vertically . The position of the muscle tached to the chitin which was held securely to the chamber was always readjusted so that the same back of the chamber with a stainless steel pressure area of the fiber was measured at the various degrees clip. The distal tendon was cut free of the exoskeleton of stretch . Tension changes resulting from stretch or and attached to a fine stainless steel wire . After the fixation were recorded on an ink-pen polygraph chitin bridge connecting the ischiopodite to the (Grass Instrument Co., Model 7) . carpopodite was removed, the glass front of the chamber was fitted into place and the chamber was Preparation for Electron Microscopy mounted vertically in the optical diffractometer. The wire on the muscle tendon was attached to the strain After single fibers had been isolated, the sarcomere transducer through an opening in the top of the lengths were adjusted to a definite length (9.6 µ, chamber . about 1 .18 X Lo) and the diffraction patterns were The preparation of the skinned single fibers was recorded . The control saline solution was replaced by similar to that described by Reuben et al . (31) . After a similar solution which contained 1 mg/ml procaine the dissection in control saline solution, the fibers hydrochloride as a means of preventing contraction were equilibrated for 30 min in a solution containing of the fiber when cold fixing medium (1 % Os04 sub- 200 mm/liter potassium propionate, 5 mm/liter stituted for an equal osmolar amount of NaCl in the MgCl2, 9 mm/liter K2EGTA, 1 mm/liter calcium control saline, pH 7 .4) was perfused through the propionate, 1 mm/liter ATP and buffered to pH 7 chamber for 3 min . A final record was made of the with 20 mm Tris . The sarcolemma was then stripped diffraction pattern before the fiber was removed from back along most of the fiber length. the chamber and placed in a cold solution of 1 % APRIL, BRANDT, AND ELLIOTT Myoltansent Lattice . 1 73 FIGURE 1 FIGURE 1 Light-diffraction patterns from a single muscle fiber stretched to various sarcomere lengths (indicated in microns) . Four orders of diffraction are visible on either side of the incident spot . The sarcomere length is inversely proportional to the distance between any order and the incident spot . The specimen-to-film distance is approximately 7 .1 cm and the photographs are reproduced at approximately X 75 . FIGURE 2 Electron micrographs and corresponding optical transforms of the cross-sectional lattice of single fibers fixed at three different sarcomere lengths : Fig . 2 a, 6.8 .s; Fig . 2 b, 8 .9 µ; and Fig. 2 c, 12 .3 µ. The fixation is with 1%Jo Os04 in the control saline solution. The electron micrographs are printed here at a magnification of X 59,000 ; however, the optical transforms were obtained from similar plates of the same fibers at about X 3000 with a specimen-to-film distance of 1 m . The distance between the first order diffraction points and the incident spot is an inverse function of the thick myofilament spacing. Os04 and Veronal acetate at pH 7 .4 (30) and fixed the incident spot is inversely proportional to the for an additional 15 min. The Epon embedding and distance between the 1, b lattice planes (dï, 0) from electron microscopic procedures were those described which the myosin interfilament distance (d.--) can by Brandt et al. (4) . The thin cross-sections were ob- be determined according to the following relation : tained from the same portion of the fiber used pre- viously in the determination of the sarcomere length di, 0/sin 60° = d,,,_,,, by light diffraction, and the specimens were carefully oriented so as to ensure that the sections were normal The accuracy of the determination of the myosin- to the fiber axis . to-myosin spacing is greatly improved by this method since the myofilament distances within one myofibril (about I µ in diameter) are averaged together . Com- Measurement of the Lattice Volumes pression effects incurred during sectioning are mini- of Fixed Fibers mized by averaging the first order diffractions in all three lattice planes .
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