(Sandow, 1952; Botts, 1957; Shanes, 1958; Bianchi & Shanes, 1959; Frank, inadequate activation of the contractile mechanism, it must be assumed

Transcription

1 110 J. Phy8iol. (1964), 170, pp With 2 plate8 and 3 text-figure8 Printed in Great Britain THE MAXIMUM SARCOMERE LENGTH FOR CONTRACTION OF ISOLATED MYOFIBRILS BY R. J. PODOLSKY From the National Institute of Arthritis and Metabolic Diseases and Naval Medical Research Institute, Bethesda, Maryland, U.S.A. (Received 13 June 1963) A. F. Huxley & Peachey (1961) recently found that when fibres from the semitendinosus muscle of the frog were stretched to various degrees and then stimulated electrically, the maximum sarcomere length at which contraction occurred was close to 3-5,u. Using electron microscopy, they also found that the resting lengths of the primary and secondary myofilaments (H. E. Huxley, 1957) were independent of sarcomere length and that the two types of myofilaments would be just pulled 'out of mesh' at sarcomere lengths close to 3-5,. The agreement of the two measurements was taken as evidence that contraction in this muscle requires interaction between the primary and secondary myofilaments in regions where they overlap. This interpretation of the correlation between the maximum sarcomere length for electrically activated contraction and the sum of the resting lengths of the two types of myofilaments rests on the validity of several assumptions. One assumption is required by the fact that electrical stimulation activates the contractile mechanism only indirectly, presumably via membrane depolarization, transverse spread of activation along the sarcoplasmic reticulum, and then longitudinal spread of activation to the force generating sites (A. F. Huxley, 1957). To ascribe loss of shortening ability to a particular configuration of the myofilaments rather than to inadequate activation of the contractile mechanism, it must be assumed that the effectiveness of the activation processes following electrical stimulation is maintained when sarcomere length is increased beyond the point where contraction vanishes. This assumption can be tested by measuring the maximum sarcomere length for contraction when the contractile mechanism is activated by a process near the end of the normal sequence of events. Micro-injection experiments suggest that the terminal step in the activation process is a transient increase in the concentration of free calcium ions (Heilbrunn & Wiercinski, 1947; Niedergerke, 1955; Podolsky, 1962). This idea is also supported by other physiological (Sandow, 1952; Botts, 1957; Shanes, 1958; Bianchi & Shanes, 1959; Frank,

2 CONTRACTION OF ISOLATED MYOFIBRILS ill 1961) and biochemical (Hasselbach & Makinose, 1961; Weber & Winicur, 1961; Ebashi & Lipmann, 1962) experiments. Therefore we studied the response of isolated myofibrils (Natori, 1954) to local application of calcium ions (Podolsky & Hubert, 1961) as a function of sarcomere length. To compare the maximum sarcomere length for contraction with the maximum sarcomere length for overlap of the two types of myofilaments, it must also be assumed that the sarcomere length at which shortening ability appears to vanish (A,) is close to the sarcomere length at which contractile force disappears (A0). Since discernible shortening requires a finite contractile force, when contractile force decreases as sarcomere length is increased by stretching (Ramsey & Street, 1940), the ability to shorten will appear to vanish before the force developed by the myofilaments falls to zero, i.e. A, < Ao. Since the magnitude of the difference between A, and Ao depends, in part, on the elastic modulus of the preparation (see Discussion), an estimate of Ao can be made by comparing the maximum sarcomere length for contraction in preparations having different elastic moduli. Fortunately the elastic modulus of myofibrils at extreme sarcomere lengths is considerably smaller than that of intact fibres (Natori, 1954) and measurement of the maximum sarcomere length for calcium-activated contraction in isolated myofibrils (A,) provides data for testing this as well as the first assumption. The present study shows that AC is close to 3-65,u; as mentioned above, Huxley & Peachey's (1961) experiments with intact fibres put the value of A, at about 3 5 u. The closeness of A' to Ak is evidence for both the above assumptions and supports the idea that loss of contractility at the critical sarcomere length is due to loss of overlap of the two types of myofilaments at that length. Some of the results have already been reported briefly (Podolsky, 1963). METHODS Disection. The preparation of myofibrils was based on the procedure described by Natori (1954). One branch of the semitendinosus muscle of the frog, Rana pipiens, was mounted in a chamber containing a modified Ringer's solution at room temperature. To reduce the excitability of the fibres 90 % of the normal amount of NaCl was replaced by an osmotically equivalent amount of sucrose. The solution had the following composition (mm): NaCl, 11-5; KCI, 2-5; CaCl,128; sucrose, 207; Na2HPO4+NaH2PO4, 3-1 (ph = 7.0); tubocurarine Cl was added to a concentration of 10-' g/l. A bundle of about 5-15 fibres was dissected out of the muscle and the adhering solution was removed by blotting. The bundle was quickly laid out on a glass cover slip and covered with paraffin oil. A single fibre was teased out of the bundle with a small eye knife and the sarcolemma was peeled away over a length of about 3 mm. Transverse cuts were made to isolate a region either completely free of sarcolemma (for critical sarcomere-length and rest-length experiments) or about half with sarcolemma and half without (for elasticmodulus experiments). The preparation remained stable for min, after which period contracture generally set in.

3 112 B. J. PODOLSK Y Manipulation of the preparation. Measurements of sarcomere rest length were made with short bundles of myofibrils, f& in length, in contact with the glass cover slip on which they were prepared. When sarcomere length was controlled, a manipulation device was used. This device consisted of two jewelers forceps (Dumont No. 5) mounted on mechanical microscope stages attached to a brass plate. The opening of the forceps tips and the height of the tips above the plate were both controlled by screw adjustments. The glass cover slip with the preparation was seated over an opening in the centre of the plate and the ends of the preparation were gripped by the forceps. The preparation was then raised clear of the cover slip, so that it was completely surrounded by oil. Sarcomere length could be varied by relative motion of the mechanical stages. Micro8copy. An inverted microscope with a binocular tube was used to observe preparations in the manipulation device. The brass plate was seated on the microscope stage and the space between the final lens of the condenser and the preparation was filled with paraffin oil. Two methods of recording were used. For measurements of the critical sarcomere length an oil-immersion phase-contrast objective was used (Unitron type D.M., focal length 16 mm, n.a. 1.25). Cine-photographs were taken at 15 frames/sec on Kodak Tri X fihm with a 16 mm camera positioned over one of the oculars; the over-all magnification on the film was 112 times. Illumination was provided by a low voltage tungsten lamp used with a water filter. The average sarcomere length was measured with an error estimated to be less than 1 % by projecting the film on to a ground-glass plate fitted with moveable cross-hairs (Vanguard Motion Analyzer, 12 x magnification) calibrated against frames of 10,u grid. The measurement was made on the frame immediately preceding application of the test solution and was taken as 1/10 of the distance between eleven striations centred about the eventual contact point of the micropipette. In one experiment ( ) averages were taken over groups of five sarcomeres in different regions of the preparation. For measurements of the contribution of the sarcolemma to the elastic modulus, where a larger field was desired, a dry phase-contrast objective was used (Unitron type D.M., focal length 4-0 mm, n.a. 0 65). Fibre diameter and sarcomere length were measured with a standard filar micrometer. For measurements of sarcomere rest length a conventional microscope was used with a narrow illuminating cone and an oil-immersion objective (Bausch and Lomb, focal length 4-3 mm, n.a. 1-00, working distance 2-5 mm; or Koristka, focal length 5-7 mm, n.a. 1-00, working distance 3-0 mm). Sarcomere length was calculated from the number of striations in the field of a calibrated eyepiece micrometer. Activation. Calcium solution was applied through glass micropipettes, coated with silicone (Desicote, Beckmann Instrument Co.), having a tip diameter of about 1 H. The composition of the solution was 140 mm NaCl+0-1 mm CaCl2. In control experiments the CaCl2 was omitted. Solutions were drawn into the tip of the micropipette by capillary action. The loaded pipettes were connected with polyethylene tubing to a micrometer-driven 1 ml. syringe; both the tubing and the syringe were water-filled. To eject solution the column of air in the shank end of the micropipette was compressed. The length of the myofibril preparation was at least 2-5 mm; the length activated by the calcium solution was less than 25 1e. Since sarcomere shortening took place in less than 1 % of the length of the myofibrils, contraction was essentially isotonic, even though the preparation was mounted isometrically. RESULTS Critical sarcomere length The ability of myofibrils to shorten in response to the application of calcium solutions was tested at various sarcomere lengths. Calcium was applied either by bringing a micropipette with a positive-pressure difference

4 CONTRACTION OF ISOLATED MYOFIBRILS 113 across the tip into contact with the myofibrils or by touching the myofibrils with drops formed at the micropipette tip. The conditions of application were such that there was no detectable response at sarcomere lengths in the range between 2 3 and 2 8,u unless calcium was present in the solution. Photographs from a typical experiment are shown in P1. 1. For each sarcomere length the frame on the left shows the band pattern just before * *0 0_ o ** Sarcomere length (u) Text-fig. 1. Results of local application of 140 mr-nacl+0.1 mm-cacl2 to myofibrils. 0, vigorous contraction; 0, no contraction; (, weak contraction. Abscissa, sarcomere length immediately before application of solution, measured from cine film; four experiments, all on fibres from the semitendinosus muscle. The numbers correspond to different preparation lengths and indicate the order in which the contractility tests were made. After each adjustment of length tests were carried out close to the same region of the preparation. The two points for run 3 of experiment are from different regions in the same field. In experiments of , and tests at successive lengths were made at different places along the length of the preparation; on tests at successive lengths were made close to the same place. calcium was applied to the fibre; the frame on the right is about 04 sec later. At an initial sarcomere length of 3-45,u calcium elicited vigorous local shortening (P1. 1, figs. 1 and 2). The pipette was in contact with the myofibrils for about 0O8 sec, during which time contraction was maintained; relaxation took place after the pipette was withdrawn and was essentially complete in less than 1 sec. When the sarcomeres were stretched to a length of 3.77 It no response was elicited by calcium ions (P1. 1, figs. 3 and 4). Contractility was restored when the same region was allowed to shorten passively to a sarcomere length of 3-48 IL (P1. 1, figs. 5 and 6). 8 Physiol. 170

5 114 R. J. PODOLSKY The results of four such experiments are shown in Text-fig. 1. Shortening occurred when sarcomere length was less than 3*6,u; shortening did not occur at sarcomere lengths exceeding 3*7,u. In one run (3 of ), the first application of calcium elicited no response; in the second and third applications one region contracted slightly while another exposed to the calcium solution still failed to respond (P1. 2). The average initial striation spacing in the contracting region was 3-64,u (standard deviation 001,u); the corresponding measurement near the centre of the exposed unresponsive region was 3-71, (standard deviation 001 Lu) (Table 1). It TABLE 1. Measurements of striation spacing for run 3 of experiment (P1. 2). Calcium solution applied three times to preparation at constant length. Data taken from the frames on original cine record immediately preceding contact of micropipette and preparation (P1. 2, top row). Frames projected on to ground glass of motion analyser and co-ordinates of eight consecutive Z lines (z,l Z2... I Z7, Z8) measured along two lines parallel to edge of preparation, one 5-6 u from edge (where contractility was maximum) and the other 3 2 & from edge (close to the centre of the region exposed to calcium but unresponsive). Z lines 4 and 5 straddled the eventual contact point of the micropipette. Individual measurements on the projected image could be read to 25 IA, which corresponded to I at the preparation. Values for Z line co-ordinates (zi) taken as the average of at least four measurements. All calculations carried to at least one extra decimal place before rounding. 3-2 z from edge 5-6,u from edge A_ A Test no Mean sarcomere length (Lt) (Z6-Z1)/ * *64 (Z7-Z2)/ (z8-z,)/ * * *64 Mean of means Standard deviation 0*01 0*01 seems reasonable to conclude that the critical sarcomere length for shortening of isolated myofibrils is close to 3-65,u (limits of error P). After the preparation of was stretched beyond the critical sarcomere length the second time (run 7), the regular pattern of striations degenerated in some parts of the myofibrils leaving localized amorphous regions. Application of calcium elicited sluggish contraction in the amorphous region, even though the remaining striated region was unresponsive. The possibility that similar degeneration of myofibril structure might occur after intact fibres are extensively and repeatedly stretched stresses the need for continuous photomicrographic monitoring in studies of the relation between sarcomere length and contractility, especially when an integrated response is used to measure contractility.

6 CONTRACTION Of ISOLATED MYOFIBRILS 115 Contribution of the sarcolemma to the elastic modulus Natori (1954) found that at lengths below 1-4 times resting length the compliance of a length of toad fibre was the same with and without the surrounding sarcolemma; at lengths exceeding 14 times resting length, the compliance of regions enclosed by the sarcolemma was less than that of regions where the sarcolemma had been removed. Sarcomere lengths were not measured. Since resting length depends partly on the way the preparation is supported (see below), we measured the average sarcomere 4.5 X A B C D 4-0 -i 0 boi I I I 2. 5p C Text-fig. 2. Striation spacing in regions of fibre with and without saxcolenuma at vaxious total lengths. The numbers on the abscissa indicate the order in which length changes were made. Ordinate, sarcomere length, measured with filar micrometer; closed symbols, region with saxcolemma; open symbols, region without sarcolemma; vertical linle through points join duplicate measurements. Fibre diameter measured at the saxcomere length corresponding to position 1 on the abscissa (in microns): A ( ) not recorded; B ( ) 58; C ( ) 82; D ( ) 77. Ratio of diamneter of region with sacolemma to region without sarcolemma < length in regions of fibres with and without sarcolemma in preparations surrounded completely by paraffin oil and supported only at the ends. The diameter of the stripped region was kept close to 'that of the unstripped region by removing the sarcolemma with as few of the adjacent myofibrils as possible; preparations were discarded when the difference i the diameters of the two regions exrceeded 7 %/. Text-figure 2 presents data from four exrperiments. Each point on the abscissa represents a different setting of the manipulator; the numbers indicate the order in which length changes were made. When the line joining two points has a positive slope, the preparation was stretched to

7 116 B. J. PODOLSKY reach the second point; when the line has a negative slope, the preparation shortened passively to reach the second point. At each setting of the manipulator, the average length of ten sarcomeres was measured. Sarcomere counts were made alternately in stripped (open symbols) and unstripped (solid symbols) regions; the variation for two measurements made in different parts of the same region is indicated by the vertical lines through the points. The experiment was terminated when sarcomeres began to contract spontaneously. The onset of contraction, which appeared min after dissection and spread slowly along the length of the stripped region, was detected by a drift of striations in the optical field. Before this contracture set in no time-dependent effects were observed. In experiment A the sarcomere lengths in both regions of the fibre were restricted to values less than about 3-3,u. The compliance is practically the same for regions with and without sarcolemma and the changes in sarcomere length are reversible. In experiments B, C and D a greater range of sarcomere lengths was used. When the sarcomere length exceeds values close to 3x2 p,, the compliance of the stripped region is always greater than that of the unstripped region. (In experiment B, when the length of the preparation was increased in moving from positions 2 to 3, the register of the myofibrils in the stripped region appeared to break down slightly; this is probably the reason for the incomplete reversibility when the length was reduced in passing from positions 3 to 4.) These observations indicate that (a) the sarcolemma resists strain more than the enclosed myofibrils at sarcomere lengths exceeding 3*2 It, and (b) the sarcolemma is linked to the enclosed myofibrils by structures capable of transmitting force. The contribution of the sarcolemma to the elastic modulus of a fibre was calculated from Text-fig. 3 (data from 10 preparations). The interrupted line denotes unit slope; the solid line was fitted by the method of least squares to the points with abscissae greater than 3-37,u. The slope of the curve formed by the data is the ratio of the elastic modulus of isolated myofibrils to that of the intact fibre. For sarcomere lengths less than 3-2,u the data fall close to the line of unit slope, indicating again that the sarcolemma does not contribute significantly to the elastic modulus at these sarcomere lengths. The tendency of the data to fall slightly below the interrupted line can probably be attributed to the fact that a few peripheral myofibrils are usually carried along with the sarcolemma when it is dissected away, reducing the cross-sectional area of the stripped region relative to that of the unstripped region of the fibre. For sarcomere lengths greater than 3*2 P the best fit is a line of slope 0-22 (standard deviation 0 056). Since the critical

8 CONTRACTION OF ISOLATED MYOFIBRILS 117 sarcomere length is in this range, the elastic modulus of the contractile system at sarcomere lengths near the critical value is reduced about fivefold by removal of the sarcolemma. The structures in the fibre which transmit to the myofibrils the force generated by the sarcolemma at sarcomere lengths exceeding 3-2 p, would ~3.5 EU E IV 0 EU u KY -,,, Sarcomere length without sarcolemma (,a) Text-fig. 3. Striation spacing in regions of fibre with and without sarcolemma. Abscissa, sarcomere length without sarcolernma; ordinate, sar omere length with sarcolemma; both measured with filar micrometer. Lines through points join duplicate measurements. Ten preparations; symbols A, *, *, and 40o are data from preparations A, B, C, and D of Text-fig. 2; data from six additional preparations denoted by +. Range of diameters (measured at sarcomere length 3 Iu) p. Ratio of diameter of region with sarcolemma to region without sarcolemma < Equation of interrupted line: y = x. Equation of solid line, derived by the method of least squares (neglecting the uncertainty in x relative to that in y since the slope <1; Deming, 1943), y = x, standard deviation of slope = be exrpected to have an elastic limit; when strained beyond this limit, the myofibrils in the unstripped region close to the stripped region should slip through the sarcolemma, like a finlger out of a glove. To see when slipping starts to occur., a particle of graphite was placed on the sarcolemnma about 20,u away from the stripped region and the fibre was slowly stretched until the striations moved relative to the marker. Myofibrils began to move through the sarcolemma when the sarcomere length reached about 4-5,u (range, p; four exrperiments with fibres of diameter ,u at sarcomere length 3,m); the sarcomere length in the stripped region was

9 118 R. J. PODOLSK Y then close to 6,. The sarcomere lengths at which slipping starts to be noticeable are clearly greater than those used in the experiments shown in Text-figs. 2 and 3. Resting length of isolated myofibrils A fibre covered with oil is surrounded by a thin aqueous layer which can adhere to a glass surface. If the fibre is extended along a glass surface, this layer tends to bind the fibre to the glass and to oppose shortening due to passive tension. When a length of myofibrils was separated from a fibre by transverse cuts, the isolated region generally shortened passively. The reason for this is probably that the magnitude of the force binding the preparation to the glass decreases when the total length of the preparation decreases, while the passive tension depends only on sarcomere length. The average sarcomere length assumed by bundles of myofibrils ,u in diameter and ,u in length was 195 (twelve preparations, range 1* p,, standard deviation 0-08,). Seven of the twelve observations were made on glass cover slips treated with a silicone preparation (Siliclad, Clay-Adams Co.) to prevent the aqueous layer from wetting the glass; the average sarcomere length for these cases was no different from that of the others. DISCUSSION The primary result of these experiments is that isolated myofibrils lose the ability to shorten in response to local application of calcium ions at sarcomere lengths close to 3-65,u. This is in reasonably good agreement with the value near 3-5 u found by A. F. Huxley & Peachey (1961) in electrically stimulated fibres from the same kind of muscle (a possible explanation for the difference is discussed below). It is also close to the value of 3.57 /pk found by Guld & Sten-Knudsen (1960) for the extrapolation to zero tension of the linear part of the twitch-tension length curve of the frog m. ext. long. dig. IV. The possibility that both the latter results are due simply to an influence of sarcomere length on a step in the activation process (as would be the case, for example, if longitudinal spread of activation from the Z line (A. F. Huxley, 1957) were limited to a distance of 1,) would be excluded by the present experiments if the terminal step in the physiological activation process is an increase in local concentration of calcium ions. The observation that the sarcolemma begins to contribute significantly to the elastic modulus at a sarcomere length close to 3*2,u would agree with the value of 14 times resting length reported for toad fibres by Natori (1954) if resting length were 2*3,u. This is somewhat greater than our value of '08, for the sarcomere length at rest, possibly because

10 CONTRACTION OF ISOLATED MYOFIBRILS 119 the total length of our myofibril preparations was shorter than that used by Natori, or perhaps because of species differences. Casella (1951), comparing the elastic properties of the intact fibre in Ringer's solution with that of the empty sarcolemma tube, concluded that the sarcolemma does not contribute to the elastic modulus until the intact fibre is stretched to 1-5 times the resting length. This is in close agreement with the present measurements and suggests that fibres immersed in paraffin oil have substantially the same elastic properties as those bathed in normal Ringer's solution. Removal of the sarcolemma from a fibre leaves a preparation in which the contractile mechanism would be expected to be unchanged, but the elastic modulus near the critical sarcomere length is reduced about five times. Nevertheless, the length at which the ability to shorten vanishes in the isolated myofibrils is no more than 02, greater than the corresponding end point in intact fibres. If the smallest detectable contraction is the same in both preparations, the closeness of these two end-points is evidence that the end point for shortening is close to the sarcomere length at which contractile force vanishes. To show this, let: P(A) = contractile force developed by a sarcomere at length A, A0 = sarcomere length at which P= 0, Ac= sarcomere length at which ability to shorten appears to vanish in intact fibres, AC = sarcomere length at which ability to shorten appears to vanish in isolated myofibrils, e = elastic modulus of intact fibre at Ac, El = elastic modulus of isolated myofibrils at Ac, Ax = smallest detectable contraction, 6 = P(Ac)/(Ao-A6), the magnitude of the slope of P(A) for A > A6. When a strained system shortens, the decrease in elastic force per unit displacement is the elastic modulus. If both intact fibres and myofibrils near the critical sarcomere length are considered to be elastic systems with a force generator acting in parallel, they will not appear to shorten actively from a given length under constant load unless the magnitude of the generated force compensates for the decrease in elastic force associated with the smallest detectable contraction. The ability to shorten isotonically will disappear in fibres whenever the contractile force is less than eax; it will disappear in isolated myofibrils for contractile forces less than e'ax. Therefore P(A6) = EAx and P(Ac) = e'ax. The magnitude of P(A) for A near A is difficult to extract from conventional isometric experiments with single fibres, since when sarcomere length in the main part of

11 120 R. J. PODOLSKY the fibre is near Ac, the sarcomere length near the ends is substantially lower (Huxley & Peachey, 1961; Carlsen, Knappeis & Buchthal, 1961). However, it is reasonable to suppose that P(A), and therefore e, will be the same for calcium-activated myofibrils and electrically activated fibres. Then, if Ax is equal in isotonic experiments with both myofibrils and intact fibres, the definition of 6 leads to the relations and.a0-a~_e - AO e (1) A07Ac= E/E'1. (2) Since c/e' > 1, equation (1) shows that Ao is better approximated by A' than by A,; the reduced elastic modulus allows isolated myofibrils to respond to smaller contractile forces than intact fibres. For A, between 3*50 and 3*54,u (Huxley & Peachey, 1961), A, between 3*63 and 3-71 lk (Text-fig. 1), and e/e' = 5 (Text-fig. 3), equation (2) brackets the value of A0 between 3-65 and 3-75,u. This consideration is relevant to the question raised by Carlsen et al. (1961) concerning the difference between A0 and A,; to the extent that the present assumptions are valid, the upper limit for A0 is close to 3.7,u. Using electron microscopy, Huxley & Peachey (1961) found that the sum of the lengths of the primary and secondary myofilaments is close to 3-5,u. For similar measurements of filament length, Carlsen et al. (1961) reported the sum to be about 34,u, whereas S. Page and H. E. Huxley (personal communication) found a value of 3-65,t. Although the species of frog used in the present experiments (Rana pipiens) is different from that used in European laboratories, where the three latter studies were made (R. temporaria), the myofilament dimensions appear to be the same. In electron micrographs of R. pipiens fibres the average length of the primary and secondary filaments were 1-5 and 1-95,u respectively, summing to 3-45,u (sarcomere lengths ranged from 2-2 to 32,u, buffered glutaraldehyde fixation, acetone dehydration, Araldite embedding, sections cut parallel to the fibre axis; E. H. Sonnenblick & D. Spiro, personal communication). Allowing for the fact that these values were not corrected for shrinkage during embedding, which can be as much as 10 % (Huxley & Peachey, 1961), the measurements suggest that myofilament dimensions are the same in the two species. Huxley & Peachey (1961) pointed out that the agreement between their measurement of Ac and the sum of the lengths of the primary and secondary myofilaments was evidence that contraction depends on interaction of the two types of myofilaments in regions where they overlap. However,

12 CONTRACTION OF ISOLATED MYOFIBRILS 121 since contractile force can be developed without discernible shortening, it is more meaningful to compare myofilament dimensions with A0, the sarcomere length at which contractile force can no longer be developed. The above analysis suggests that A0 is between 3-65 and 3-75 u, which is still in good agreement with the sum of myofilament lengths, especially the values found by S. Page and H. E. Huxley (personal communication). Although this provides additional support for the view that contraction in these fibres requires overlap of primary and secondary myofilaments, the question of how contractile force is generated in the first place, and particularly whether overlap is required for force development per se or simply for the transmission of force developed in a single myofilament, is still not clear. It should be noted that the sarcomere length of isolated myofibrils at rest ( ,u) is close to the length of the secondary myofilaments (19 p, Huxley & Peachey, 1961; 18,u, Carlsen et al. 1961; 205,u, S. Page and H. E. Huxley, personal communication), which suggests that resting length may be determined by end-to-end abutment of these filaments. Another observation that bears on the arrangement of the myofilaments is the fact that loss of ability to shorten at extreme sarcomere lengths can be reversed simply by allowing stretched myofibrils to shorten passively. This is consistent with the suggestion that the ends of consecutive secondary filaments are connected to each other through the H zone (Hanson & H. E. Huxley, 1955), for otherwise it would be difficult to see how, after being withdrawn, the secondary filaments re-enter the array of primary filaments without tangling. Since it is absent, the sarcolemma clearly is not involved in the restoration of contractility. SUMMARY 1. Myofibrils isolated in paraffin oil from striated muscle fibres of the frog were tested for the ability to shorten in response to local application of calcium ions. 2. Shortening followed by relaxation occurred when the sarcomere length was less than 3-6,. Shortening did not occur at sarcomere lengths exceeding 3 7 u. The disappearance of the ability to shorten was reversible. 3. Passive tension in myofibrils becomes zero at sarcomere lengths close to 19,u. The sarcolemma does not appear to contribute to passive tension at sarcomere lengths less than 32,u; beyond this length the sarcolemma resists strain more than the enclosed myofibrils. The sarcolemma is mechanically coupled to the enclosed myofibrils. At sarcomere lengths exceeding 3*2, the elastic modulus of a fibre is reduced about fivefold upon removal of the sarcolemma. 4. The close agreement between (1) the maximum sarcomere length

13 122 R. J. PODOLSKY for shortening in calcium-activated contraction of isolated myofibrils and (2) the maximum sarcomere length for shortening in electrically activated fibres is evidence that the normal activation processes are not significantly affected by sarcomere length. The magnitude of the difference between (1) and (2) suggests that the sarcomere length at which the contractile force becomes zero is less than 3-75,u. 5. These observations are evidence that a critical change takes place in the contractile mechanism rather than in the activation mechanism at a sarcomere length between 3-6 and 37,u. Since the sum of the lengths of the primary and secondary myofilaments is also close to this value, this provides additional support for the view that contraction requires overlap of the two types of myofilaments. The author is indebted to Mr R. D. Arrieta for invaluable technical assistance throughout the course of these experiments. The views expressed in this article are those of the author and do not necessarily reflect the opinions of the Navy Department or the Naval Service at large. REFERENCES BLNCHI, C. P. & SHEANs, A. M. (1959). Calcium influx in skeletal muscle at rest, during activity, and during potassium contracture. J. gen.. Phy&iol. 42, Borrs, J. (1957). Triggering of contraction in skeletal muscle. In Phy8iological Trigger8, ed. BuLLocK, T. H., pp Washington: Amer. Physiol. Soc. CARLSEN, F., KwAPPEIs, G. G. & BucETHAL, F. (1961). Ultrastructure of the resting and contracted striated muscle fiber at different degrees of stretch. J. biophy8. biochem. Cytol. 11, CASELLA, C. (1951). Tensile force in striated muscle, isolated fiber and sarcolemma. Acta physiol. 8cand. 21, DEMING, W. E. (1948). Statistical Adjwdtment of Data. New York: Wiley. EBASm, S. & LIPMANN, F. (1962). Adenosine triphosphate-linked concentration of calcium ions in a particulate fraction of rabbit muscle. J. cell. Biol. 14, FRA&NK, G. B. (1961). Role of extracellular calcium in excitation-contraction coupling in skeletal muscle. In Biophysics of Physiological and Pharrwcological Actions, pp Washington: Amer. Ass. Adv. Sci. GuD, C. & STEN-KNuDsEEN, 0. (1960). Correlation of isometric twitch tension and latency relaxation to the sarcomere length in frog muscle fibers. Acta physiol. 8cand. 50, Suppl. 175, HANSON, J. & Hux;xY, H. E. (1955). The structural basis of contraction in striated muscle. Symp. Soc. Exp. Biol. 9, HASSELBACH, W. & MAKINoSE, M. (1961). Die Calciumpuampe der 'Erschlaffungsgrana' des Muskels und ihre Abhangigkeit von der ATP-Spaltung. Biochem. Z. 333, HEILBRUNN, L. V. & WIERcrNsKI, F. J. (1947). The action of various cations on muscle protoplasm. J. cell. comp. Physiol. 29, HuxLEY, A. F. (1957). Muscle structure and theories of contraction, pp in Progress in Biophysics, vol. 7, ed. BUTLER, J. A. V. and KATZ, B. London: Pergamon Press. HuXLEy, A. F. & PEACHEY, L. D. (1961). The maximum length for contraction in vertebrate striated muscle. J. Physiol. 156, HuxLEY, H. E. (1957). The double array of filaments in cross-striated muscle. J. biochem. biophy8. Cytol. 3, NATORI, R. (1954). The property and contraction process of isolated myofibrils. Jikeikai nmd. J. 1, NWEDERGERKE, R. (1955). Local muscular shortening by intracellularly applied calcium. J. Phy8iol. 128, P.

14 The Journal of Physiology, Vol. 170, NYo. 1 Plate I I m co (ri) 432ual a-lawodies R. J. PODOLSKY( (Facit7g p. 122)

15 The Journal of Physiology, Vol. 170, No. 1 Plate 2 I I R. J. PODOLSKY

16 CONTRACTION OF ISOLATED MYOFIBRILS 123 PODOLSKY, R. J. (1962). The structural changes in isolated myofibrils during calcium activated contraction. J. gen. Physiol. 45, 613A-614A. PODOLSKY, R. J. (1963). The maximum sarcomere length for calcium activated contractions in frog myofibrils. Abstr. Biophys. Soc. MD 8. PODOLSKY, R. J. & HUBERT, C. E. (1961). Activation of the contractile mechanism in isolated myofibrils. Fed. Proc. 20, 301. RAMSEY, R. W. & STREET, S. F. (1940). The isometric length-tension diagram of isolated skeletal muscle fibers of the frog. J. cell. comp. Physiol. 15, SANDOW, A. (1952). Excitation-contraction coupling in muscular response. Yale J. Biol. Med. 25, SHANES, A. M. (1958). Electrochemical aspects of physiological and pharmacological action in excitable cells. Pharmacol. Rev. 10, WEBER, A. & WiNIcuR, S. (1961). The role of calcium in the super-precipitation of actomyosin. J. biol. Chem. 236, EXPLANATION OF PLATES PLATE I Frames from run of , all from the same region of the preparation. Figures on the left immediately before micropipette containing calcium solution touched the myofibrils; figures on the right about 0 4 sec later. Stage micrometer 10 1e divisions. Figs. 1 and 2. Initial sarcomere length, 3-45 p; contraction is vigorous. Figs. 3 and 4. Sarcomere length increased to 3-77,u; contractility appears to vanish. Figs. 5 and 6. Sarcomere length reduced to 3-48,u; contractility restored. PLATE 2 Frames from run 3 of , all of same field and with preparation held at constant length. Three tests for contractility; in each column top print is frame immediately before micropipette containing calcium solution touched preparation, centre print shows maximum reach of micropipette, and lower print is about 1 sec after initial contact of micropipette. Micropipette completely withdrawn from preparation within 1-5 sec after initial contact; after withdrawal, about 1-5 sec passed before beginning of next test. Average striation spacing on frames in top row near eventual contact point of micropipette: 3-2, from edge, 3-71,u; 5-6 u from edge, 3-64 I (Table 1). Stage micrometer, 10,u divisions. Figs Test 1: no detectable contraction. Fig. 1, before exposure to test solution; fig. 2, 0-3 sec later, pipette at maximum reach, contact length of preparation along tip of micropipette is 8 u; fig. 3, 1 0 sec after initial contact of pipette, no detectable change in configuration of striation pattem. Figs Test 2: slight contraction at about 6 u from edge, region closer to edge exposed to test solution but is unresponsive. Fig. 4, before exposure to test solution; fig. 5, 0 3 sec later, pipette at maximum reach, contact length of preparation along tip of micropipette is 9,u; fig. 6, 1x2 sec after initial contact, striations marked by arrow are displaced relative to initial configuration in region about 6 u from edge, in region closer to edge no contraction is detectable. Figs Test 3: results similar to test 2. Fig. 7, before exposure to test solution; fig. 8, 0-3 sec later, pipette at maximum reach, contact length of preparation along tip of micropipette is 9 u; fig. 9, 0-9 sec after initial contact, striations marked by arrow are displaced relative to initial configuration in region about 6 p from edge, in region closer to edge no contraction is detectable.