177

J. Physiol. (1979), 292, pp. 177-192 With 2 plates and 8 text-figures Printed in Great Britain

SARCOMERE LENGTH-TENSION RELATIONS OF FROG SKINNED MUSCLE FIBRES DURING CALCIUM ACTIVATION AT SHORT LENGTHS

BY R. L. MOSS Research, Boston Biomedical Research Institute, of Muscle From the Department Boston, Massachusetts 02114, U.S.A.

(Received 27 July 1978) SUMMARY

1. Single twitch fibres were isolated from anterior tibial muscles of the frog, Rana pipiens. The relationship between sarcomere length and steady tetanic tension at 5 TC was obtained from these living fibres in the range of sarcomere lengths between about 2-2 and 1*3 #am. These fibres were then either mechanically or chemically skinned. 2. Segments were cut from the skinned fibres and mounted in an experimental chamber using a technique designed to minimize segment compliance at the points of attachment. A piece approximately 1 mm in length remained exposed to the bathing solution. 3. The segments were photographed through a light microscope at magnifications of about 460 or 110 x during activation and relaxation, so that the sarcomere lengths could be determined from a part or the whole of the segment. Activations were done with solutions of pCa either 5'49 or 6*09 and at a temperature of 5 'C. Fibre segments which developed striation pattern irregularities during contraction were rejected. 4. The sarcomere length-tension relation obtained from these segments in the sarcomere length range 1-3-2-2 ,tm was similar to that obtained from the same fibres while still living. The results were similar at the two values of pCa used. 5. These results do not support the view that sarcomere length dependent variation in the amount of calcium which is released during tetanic stimulation is a major determinant of the form of the length-tension relation in living muscle fibres at sarcomere lengths less than about 2*0 ,im. INTRODUCTION

The relation between isometric tension and sarcomere length in intact single muscle fibres from the frog was established by Gordon, Huxley & Julian (1966a, b). They observed a plateau in the tetanic tension between sarcomere lengths of about 2-0 and 2*2 ,um. For lengths greater than about 2-2 ,tm (the descending limb of the length-tension relation), tension declined linearly as sarcomere length was increased. At lengths below 2*0 jtm (the ascending limb), the steady tension declined linearly to about 1 7 #um (the shallow part of the ascending limb), with a slope less than that of the descending limb. At 1*7 /tm, the slope of the length-tension relation increased 0022-3751/79/3980-0641 $01.50 © 1979 The Physiological Society

R. L. MOSS sharply (the steep part of the ascending limb) and tension declined linearly to zero at a sarcomere length of about 1-3 jsm. The continuous lines in Fig. 8 represent the plateau and ascending limb. Gordon, Huxley & Julian (1966a, b) interpreted their results in terms of mechanical and structural factors at the level of the sarcomere. Variation in the amount of thick and thin filament overlap as sarcomere length was altered formed the basis for the explanation of the plateau and the descending limb. Interference of thin filaments with cross-bridges in the opposite half of the sarcomere and the meeting of the thin filaments in the middle of the sarcomere, as well as the possible presence of substantial hydrostatic pressures, were suggested as determinants of the shallow part of the ascending limb; and the progressive compression of the ends of the thick filaments against the Z-disks was thought to explain the steep part. More recently, it has been suggested that at short lengths there is a possible influence of sarcomere length on excitation-contraction coupling (Taylor & RUdel, 1970; Riudel, & Taylor, 1971). In particular, the amount of calcium which is released during tetanic stimulation may vary as the sarcomere length is altered, possibly as a result of a partial failure of the inward spread of activation. The present study investigates the form of the ascending limb of the lengthtension relation in chemically and mechanically skinned muscle fibre preparations, in which the calcium concentration of the solution bathing the myofibrils can be directly controlled. Prior to skinning, the ascending limb was determined for the living fibres during tetanic stimulation. Techniques were used which permitted direct measurement of sarcomere length in both the living and skinned fibres at rest and during steady contractions. The results in general do not differ from those reported earlier by Gordon, Huxley & Julian (1966a, b). This indicates that, at least for tetanic contractions at about 5 'C, length-dependent variation in calcium release is not a major determinant of the shape of the ascending limb of the length-tension relation in vertebrate skeletal muscle. Brief reports of these results have previously been made to the American Biophysical Society (Moss, Sollins & Julian, 1977; Moss & Julian, 1978).

178

METHODS

Preparation Isolation of fibre8. Anterior tibial muscles were dissected from the legs of either winter or summer Vermont frogs (R. pipien8) and placed in Ringer solution containing (mM): Nacl, 115; K11, 2*5; CaCl2, 1-8; Na2HPO4, 2-15; NaH2P04, 0-85; pH 7-2. Single muscle fibres were then isolated from the medial head using 27 gauge needles and knives broken from razor blades. The fibres were selected to have diameters in the range 75-100 j#m in order to fit into the connectors which were used to attach skinned fibre segments to the experimental apparatus. Fibre diameter increased by about 20 % when the fibres were either briefly glycerinated or mechanically skinned, using the procedures described below. Solution and protocol for chemical treatments. The solutions which were used for the brief glycerination procedure, which will be denoted 'chemical skinning', have already been described (Julian, 1971). Following the mechanical measurements which were made on most of the fibres while intact and living, fibre length was adjusted to the just taut length, corresponding to a sarcomere length of about 2-0-2-10 sum. The fibres were then depolarized in high potassium solution containing 100 mm-KCl, 2 mM-EGTA, and 10 mm-phosphate buffer (pH 7.0) at room

LENGTH-TENSION RELATIONS OF SKINNED MUSCLE

179

Fig. 1. Connecting device for attachment of skinned fibre segments to experimental apparatus. B is the main body of the connector; M is the end of a skinned muscle fibre segment; and P is the stainless steel pin. In practice, the pin is placed down upon the segment end and secured in place with two loops of 10-0 monofilament. temperature (21-23 0). This solution was replaced with cold (about 4 'C) glycerine solution containing 47-3 % (v/v) glycerine, 2 mM-EGTA and 10 mM-phosphate buffer (pH 7.0), in which the fibres were soaked for about 60 min. The fibres were rinsed with high potassium solution, and a fibre segment was placed in a small glass spoon, containing high potassium solution, for transfer to an experimental trough. The segment was then attached horizontally between two wires, shown in Figs. 2 and 3, one extending from a force transducer and another which was firmly secured in the bath. Details of the attachment method are described in the following section. A length of segment approximately 1 mm long was left exposed to the bathing medium. The high potassium solution was then replaced with relaxing solution containing 0-5 % (w/v) of either Brij 58 or Lubrol-WX. The segment was bathed in this solution for 30 min before transfer to the standard relaxing solution. Relaxing solution was composed of the following (mM): KCl, 100; MgCl2, 1; ATP, 4; EGTA, 2; imidazole, 10; pH 7 0). A few fibres were mechanically skinned (Natori, 1954), rather than undergoing the brief glycerination described above. These fibres were first depolarized in high potassium solution and were then placed in standard relaxing solution. Mechanical skinning was done by piercing the sarcolemma at one end of the fibre with the bent tips of two 27 gauge hypodermic needles. The sarcolemma could then be rolled or slid toward the centre of the fibre. Care was taken during this procedure not to stretch unduly the skinned portion of the fibre. Segments were cut free, transferred to the experimental trough, and attached to the apparatus, all while in standard relaxing solution. The mechanically skinned segments were not soaked in the detergent relaxing solution. The chemically and mechanically skinned segments were activated in solutions containing added calcium. The calcium concentration was controlled to the desired levels using EGTA (or ethylenebis (oxyethlene-nitrilo)-tetraacetic acid), assuming the apparent stability constant for Ca-EGTA to be 106'8 as described previously (Julian, 1971). In the present experiments, pCa values of 6-09 and 5-49 were used. The lower pCa was maximally activating with regard to tension in the fibre segments. A pCa of 6-09 was often used, however, since segments activated at this pCa developed tensions which were about 90 % of maximal, and yet the segments tended to maintain better striation uniformity and could be used for many more measurements than those fibres consistently activated at pCa 5-49. The results to be described in this paper did not differ significantly at the two pCa values used. In some instances, activations were preceded by bathing the segments in a relaxing solution containing 3-5 mM-HDTA (1,6-diamino-hexane, N,N,N',N'-tetraacetic acid; Fluka Chemical Co.) and 0.5 mM-EGTA.

180

R. L. MOSS Apparatwu

Connexion to thefibre segment. Attachment of the segment ends to the two wires in the experimental trough was done by means of miniature, two-piece connectors, shown in Fig. 1. The main body of each connector was fabricated from a 1-5 mm length of thin-walled stainless steel tubing (0 30 mm o.d., 0-15 mm i.d.; Small Parts Inc., Miami, Florida). For a length of t mm along Thermoelectric devices

Lateral adjustment

Base

Fig. 2. Diagram of the top view of the experimental chamber and associated apparatus. The thermoelectric devices are mounted between the base and a water-cooled heat sink. The operation of the moveable plate to change solutions is explained in the text. this piece the metal was half ground away, leaving a long narrow trough. This piece was then cemented with Shellac to one of the wires, keeping the long axis of the piece in the horizontal position. A second similar piece was attached to the other wire. A fibre segment in high potassium solution was positioned with each of its two ends in one of the troughs. Polished, stainless steel pins (0-16 mm diameter; 0*7-0-8 mm long), the ends of which were slightly rounded, were placed in the troughs on top of the segment ends. Each pin was secured in place with two loops of 10-0 nylon monofilament suture. This attachment method was found to produce less segment end compliance than any other which was tried. The high potassium solution, which induced segment rigor during attachment, was used in order to minimize the end compliance. Mechanically skinned segments, which were mounted in the connectors while in relaxing solution, generally were more compliant. A clamp-like connecting device (Moss, Sollins & Julian, 1976) was used in some of the earlier experiments. These connectors were not satisfactory in that they more often than not damaged one of the segment ends, giving rise to high preparation compliance. Even when damage was not evident by microscopic examination, the segment ends were severely distorted by the grip of the device. Force measurement. The force transducer used, shown in Figs. 2 and 3, was a capacitance gauge, similar to that described by Julian & Sollins (1973). The present unit had a resonant frequency of 800-130 Hz, depending upon the stylus employed, and the sensitivity was adjusted to 2 mV/mg.

LENGTH-TENSION RELATIONS OF SKINNED MUSCLE

181

Capacitance changes were detected using an FM circuit similar to that of Cambridge & Haines (1959). The output from the detector circuit was recorded on a strip chart recorder (HewlettPackard, Model 7133A) and was also monitored using a storage oscilloscope. Experimental chamber. This consisted of a flat, rectangular aluminium plate, shown in Fig. 2, which had three thermo-electric devices (Cambion) mounted along one side for cooling. The centre portion of the plate was spring loaded from below, so that this piece was normally level with the remainder of the plate. A large rectangular opening cut into the movable portion,

|Eyepiece Motorif

';

IWJ k arm

oo

| transducer |

Obetv

(a) / Fibre

Thermistor

i

/

| OscillForce

amp.sexlor-

\ Conn~~~~~dector/

surface ~~~FrontMirror

P~ig. 3. Schematic diagram of experimental apparatus. The motor, objective, force transducer, fibre segment and experimental trough are drawn in approximate relative scale. The records of tension va. time on the recorder and oscilloscope are hand drawn; the arrows indicate the approximate time during contraction that photomicrographs of the segment would be taken. Inset: enlarged view of fibre segment mounted in the connectors. The strands of nylon monofilament which hold the stainless steel pins in place are not shown. See Fig. 1 for detailed view of connector. and a Plexiglass piece was fabricated to divide the opening into three troughs, each having clear floors to allow illumination from below for microscopy. The centre piece was guided by pins, so that solution changes could be done by depressing thepiece, sliding it laterally and then releasing. Three-way position translators were mounted to either side of the centre piece and allowed for adjustment of fibre position and length. The entire plate and solution-changing assembly were mounted on the modified stage of a Zeiss WL microscope. During the solution change procedure, the fibre segments twice passed through an air-fluid interface; however, observation of the segments through a microscope revealed that no damage occurred during the transfer. Micro8copy. Segments were observed and photographed through an ordinary light microscope in the manner shown in Fig. 3 using either of two lens configurations. A mercury vapor lamp was used for illumination and the field stop was focused at the specimen plane. The condenser (Zeiss model VZ) was stopped down to enhance the contrast of the striations for both viewing and photography. A magnification of about 460 x was obtained using a 40 x air objective

182

B. l. MOSS

(Zeiss, N.A. 0-65) and 10 x eyepiece (Zeiss). With this system, photographs encompassing 0-3 mm lengths of fibre were taken using Polaroid 107 film. This lens and film combination was used exclusively in the experiments involving the living fibres and in the early experiments on briefly glycerinated and mechanically skinned preparations. A magnification of about 110 x was achieved with a 10 x air objective (Zeiss, N.A. 0-22) and 5 x eyepiece (Zeiss), which were used in the later experiments involving chemically activated preparations. The eyepiece was used to focus the image on 35 mm film (Kodak High Contrast Copy film). This camera and lens combination permitted photography of an entire segment, up to 1-2 mm long, in a single frame. Photographs were taken by manually actuating the camera shutter at selected times while the segments were relaxed or during steady contractions. The developed negatives were projected on an enlarger and rows of striations were counted and measured. Where possible, three rows of striations running the entire segment length were analysed from each segment. The method of sarcomere length determination is illustrated in PI. 1.

Experimental protocol Livingfbre preparations. Living single muscle fibres were mounted in an experimental apparatus, which has been described previously (Julian & Sollins, 1975). The Ringer solution bathing the fibre was cooled to 5 0, and fibre length was adjusted so that the fibre was just taut (sarcomere length in the range 2-0-2-1 /sm). The width of each fibre was measured from a photomicrograph obtained while the fibre was at rest. The cross-sectional area of the fibre was measured as described by Gordon, et al. (1966 a). Tetanic stimulation (Julian & Sollins, 1975) was applied to the fibre, with ends held fixed, for a duration of 400-1200 msec and at a frequency of about 35 Hz. After the development of steady tetanic tension, photomicrographs of central fibre segments were taken using the high power lens configuration described above. Sarcomere lengths less than 2-0 #sm were obtained during tetanic contractions by introducing an appropriate amount of slack into the fibre, and photomicrographs were taken during the contractions. The fibre was finally returned to the just-taut length for a control tetanic contraction. At this point, the fibre was either briefly glycerinated or mechanically skinned. Skinned fibre preparations. Following skinning and attachment, the fibre segments were placed in fresh relaxing solution and cooled to 5 0C. The sarcomere length was adjusted to about 2-25 and the segment was then placed in activating solution. When a steady tension had developed, a photomicrograph was taken and the fibre was returned to the relaxing solution. The sarcomere length was measured from this photograph and the segment length was adjusted, if necessary, to give a sarcomere length of 2-15 during contraction. Once a contraction was obtained in which the average sarcomere length was between 2-10 and 2-20jam, the segment was slackened to give an estimated sarcomere length of about 2-0, 1-8, 1-6, or 1-4 ,um during activation. The segments were activated twice at the shorter lengths, before returning to the original over-all length. The first contraction of each pair was done at an estimated sarcomere length of 2-0 or 1-8 sum and the second at 1-6 or 1-4jsm. This procedure minimized the striation pattern disorganization which otherwise occurred if the fibres were allowed to shorten extensively under little or no load. The tensions measured at sarcomere lengths less than 2-15,sm were expressed as a percent of the steady tension at 2-15 tm. The decline in the maximum tension-producing ability of the segments which occurred in successive contractions was taken into account in this normalization by linear interpolation between the control values obtained at about 2-15 #am. An example of this correction procedure is shown in Fig. 4. For most of the segments which were used, the first few contractions (varying in number from 2 to 6) gave approximately constant maximum tensions at 2-15 ,am. Thereafter, maximum tension at this sarcomere length declined in nearly linear fashion, at a rate which varied from segment to segment. From one to five different sarcomere lengths below 2-15 ,um were explored in each segment. Including the initial length adjustment and the control and experimental measurements, as many as fourteen acceptable contractions as determined by the criteria below were obtained from a single segment. Preparation evaluation. In this work, the following criteria were applied to determine whether the condition of a fibre segment was acceptable for experimentation. First, after the skinning and mounting procedures, any fibre segment which showed a high degree of striation nonuniformity along its length, while in relaxing solution at a sarcomere length of 2-25jm, was not used. A variation in sarcomere length of about 0-2,tm or less was considered satisfactory for

c#m,

#cm

LENGTH-TENSION RELATIONS OF SKINNED MUSCLE

183

these experiments. Any segment which showed an obviously damaged region while relaxed or activated was discarded. Segments were also rejected if (1) the maximum Ca-activated tensions at about 2-15 jAm fell to 60 % or less of the tension which was obtained during the first contraction at this length, (2) the segment was found by microscopic observation to have an unduly compliant end connection or (3) a sudden large tension decline was observed to occur in the second of two succeeding control contractions at about 2 15 Asm. 0-2 mN

K 20 sec

A 2 15 Mm B 1 80 Mm C 2l14Mm Fig. 4. Photographs of tension records demonstrating the technique used to correct for decline in ability of fibre segment to generate tension. The three contractions in solution of pCa 5 49 were obtained in sequence from the same fibre segment. Photomicrographs of the segments were obtained when the active tension reached an approximately steady level. The average sarcomere lengths measured from the photomicrographs were: 2-15 ,m, in A; 1.80 eAm, in B; and 2X14 jsm, in C. In each case, the first arrow indicates the change from relaxing into activating solution; the second arrow indicates the change back into relaxing solution. The details of the correction for tension decline are described in the text. Segment was chemically skinned.

Tensions exerted by the relaxed fibre segments were measured following each cycle of activation and relaxation at a sarcomere length of about 2-15 /am. This was done by introducing slack into the segment and observing the change in the tension trace. The segments generally had tensions of 5 #sN or less in relaxing solution. If a tension of 20 IAN or more was measured, the segment was discarded. However, only a few segments had to be rejected for this reason, since the criteria regarding striation uniformity seemed a far more sensitive index of segment condition. RESULTS

lxngth-tension relaion in living and skinned fibres. The living muscle fibres were stimulated tetanically at a measured sarcomere length of about 2-15 Am, as shown in Fig. 5a. The maximum steady tension per unit cross-sectional area of the fibres averaged 250 kN/m2 in five fibres. The resting fibres were then slackened to achieve

R. L. MOSS 184 various shorter sarcomere lengths during contraction. Photomicrographs obtained during steady tetanic contractions indicated that sarcomere length was accurately determined by introducing an amount of slack based directly on the average sarcomere length measured at the just-taut length. This proportionality between sarcomere length and the amount of slack indicates that end-to-end variations in sarcomere length such as are found on the descending limb (Huxley & Peachey, 1961; A

.5mNL

C

05mNL

200 mseclse

B ,,,,4..,. IH D &5 mN ..0 msc

L 3 ........... f

0-5 mN L 13 sec

Fig. 5. Tension records obtained from a living fibre and a chemically skinned fibre segment. A and B are tetanic tension records obtained at measured average sarcomere lengths of 2*15 jam (A) and 1-68 jam (B). In B, the time required to take up the imposed slack would account for the brief delay in the onset of tension development. The further slow rise in tension presumably results from the relatively slow attainment of the final sarcomere length. C and D are the tension records obtained from a chemically skinned fibre segment in solution of pCa 6-09 at measured average sarcomere lengths of 2- 10 jtm (C) and 1-64 jum (D). The first arrow in both parts indicates the transfer from relaxing to activating solution, and the second arrow indicates the transfer back to relaxing solution.

Julian, Sollins & Moss, 1978) are not present in the fibres on the ascending limb. Thus, sarcomere length measurements from a central segment of the living fibre are representative of the average sarcomere length of the entire fibre. An example of a tetanic tension record obtained at an average sarcomere length of 1 68 jtm is shown in Fig. 5 B. The fibres were never shortened below a sarcomere length of about 1 3 jtm to avoid the onset of the delta-state described by Ramsey & Street (1940). The sarcomere length-tension data for the living fibres are plotted as the open circles in Fig. 6. There is good agreement between the present data and the length-tension relationship which was obtained by Gordon et al. (1966b) for tetanically stimulated frog fibres in the same sarcomere length range. The living fibres were next either briefly glycerinated or mechanically skinned, as described in the Methods. Sarcomere length-tension relations of segments cut from the skinned fibres were then measured using activating solutions of pCa 6*09. The maximum steady tensions obtained in these fibres at a sarcomere length of about 2-15 #um averaged approximately 77 % of that obtained in the same fibres while living and tetanically stimulated in the sarcomere length range 2-0-2.2 ,sm. Since pCa 6-09 produces a tension which is about 90 % of the maximum that could be induced by chemical activation, the amount of tension developed by the skinned fibre segments is comparable to that developed by the living fibres. Several activa-

185 LENGTH-TENSION RELATIONS OF SKINNED MUSCLE tions were done in solutions containing 14 mM-creatine phosphate and 1 mg/ml.

creatine phosphokinase (Godt, 1974). No increase in the maximum developed tension was seen when these solutions were used, suggesting that the supplies of ATP in the segment cores were adequate to sustain contraction. This result is not unexpected in view of the total concentrations of ATP (4 mM) and Mg2+ (1 mM) used, and also the low temperature (5 'C) of the solutions and the small diameters of the segments which were used. 1 0p

0

of0 0~~~~ 0

C

0

0-6

0

C

/

z4U 06 0D

0

0

0-2 0

0*0

0

I 1-5

I

I1

,

,a

2-1 2-3 2-5 2-7 Sarcomere length (Mm) Fig. 6. A plot of steady developed tension as a function of sarcomere length. The open circles are data points obtained from living, tetanically stimulated muscle fibres. Those fibres were then chemically or mechanically skinned. The filled circles are data points obtained from chemically skinned and the triangles from mechanically skinned segments during steady tension generation in solution of pCa 6-09. For each fibre and segment, the measured steady tensions at sarcomere lengths less than 2-0 jam were scaled to the maximum tension developed by the same preparation in the sarcomere length range 2-1-2-2 ,um. The continuous line represents part of the isometric sarcomere length-tension relation described by Gordon et al. (1966b) for tetanically stimulated frog skeletal muscle fibres at 4-6 'C. 1-3

1-7

1-9

Fig. 5C and D show tension records obtained from a briefly glycerinated segment at measured sarcomere lengths of 2- 10 jam (C) and 1-65 #sm (D). These records are characteristic of the time courses of tension rise observed in the preparations of this study in solutions of pCa 6-09. The slower rate of rise of tension relative to the living fibres at a sarcomere length of 2.10-2.20 jam presumably resulted from the mixing of the Ca2+-containing EGTA buffer system of the activating solution with the essentially Ca2+-free EGTA system initially present in the segment upon activation. This problem has already been pointed out by Moisescu (1976); he used activating solutions of high Ca-buffering capacity and relaxing solutions of relatively much lower Ca-buffering capacity to achieve rates of tension rise, in skinned fibres, which were comparable to the

R. L. MOSS

186

rates of rise reported for living fibres. A modification of Moisecsu's solutions was used in the present study for several of the tension measurements. The segments were bathed in a relaxing solution containing 3-5 mM-HDTA And 0 5 mM-EGTA, rather than the standard 2 mM-EGTA. These concentrations of HDTA and EGTA were chosen to give a total concentration equal to the concentration of EGTA in the activating solutions. The activations which followed the HDTA

1*0

08

06 0

0*4

0-2

0*00

13

17 1*9 2-1 : Sarcomere length (pm)

Fig. 7. A plot of steady developed tension as a function of sarcomere length in chemically skinned fibre segments activated in solutions of pCa 5 49. Developed tension is scaled in the manner described in the legend of Fig. 6. The continuous line is from Gordon et al. (1966 b). All data points were obtained from chemically skinned segments. soak resulted in half-times of tension rise which were nearly 10 times shorter than those measured during activations without the HDTA pre-treatment. The maximum tensions which the segments developed were about the same, whether or not the segments were pre-treated with HDTA. Paradoxically, the use of HDTA generally resulted in an increase in striation disorder during activations with high levels of free calcium. For these reasons, the relaxing solutions containing HDTA were not routinely used.

The further delay in tension rise in D relative to C in Fig. 5 is due both to the uptake of the initial slack in the fibre, and also rearrangements of length among sarcomeres which probably occur at later times following activation. The sarcomere length-tension data for the skinned preparations, activated at pCa 6-09, are shown as filled circles in Fig. 6. These data follow closely the relationship found by Gordon

LENGTH-TENSION RELATIONS OF SKINNED MUSCLE 187 et al. (1966b), at sarcomere lengths less than 2-20 ,tm, and also agree well with the sarcomere length-tension data measured in the same fibres while living and stimulated to contract tetanically. Length-tension relation in maximally adivated skinned fibres. Sarcomere lengthtension data were also obtained in several fibre segments at a pCa of 5-49. This was done in order to test whether the agreement found between the data at pCa 6-09 and that which was measured in the tetanically stimulated living fibres was a result of that particular Ca2+ concentration, or whether such agreement is a general characteristic of the length-tension relations obtained in solutions containing high free calcium concentrations. These measurements were particularly difficult to make, because comparatively few segments did not develop regions of severe striation nonuniformity when allowed to shorten below a sarcomere length of 1 .5-1-66um. Examples of contractions in solutions of this pCa are shown in Fig. 4. The data which were obtained are plotted in Fig. 7. In this case, also, there is no tendency to deviate from the length-tension relationship described by Gordon et al. (1966b). At the shortest sarcomere lengths measured, that is, less than about 1-4 #um, there is an increase in data scatter which is associated with an observed increase in the variability of the sarcomere length measurements in this length range. Length-tension relations obtained while viewing the whole of the segment. After the experiments described above were completed, the apparatus was modified to take photomicrographs of the whole skinned fibre segment, as described in the Methods. The photograph of P1. 1A is an example of one of the chemically skinned segments which was used. Once photographs were obtained, tracings such as the one in P1. 1 B were made and the sarcomere lengths were measured in various parts of the segment. A mean sarcomere length for the segment was then determined as the average of the several measurements of striation spacing which had been made. An example of the striation patterns obtained during steady activation at pCa 5-49 is shown in P1. 2. The length-tension data from six segments in which sarcomere length was measured from the length of the whole segment is shown in Fig. 8A. The activating solution had a pCa of 5-49. At overall segment lengths estimated to give sarcomere lengths less than about 1-5 ,um during activation, it was not possible to obtain striation patterns except in the centres of the segments. The maximum tensions measured at these segment lengths were therefore plotted as open circles against the sarcomere length estimated from the over-all length of the activated segment at a measured sarcomere length of about 2*15 ,tm. These data points were plotted only if the segments had no striation pattern irregularities when a control activation was done at about 2-15 msm. The filled circles in Fig. 8A are data points for which sarcomere length could be measured over most of the length of the segment. The large error associated with each point indicates the degree of striation non-uniformity associated with contractions at sarcomere lengths less than 200 ,sm in solutions of pCa 5*49. Also, these data points tend to fall to the right of the curve obtained by Gordon et al. (1966b) in living fibres. Comparison of this data to that in Fig. 7, in which only the central 03 mm of the segment was photographed, suggest that during activation the central portions of the segments shortened against the ends. Apparently, the small piece of muscle fibre which is pulled out of each connector is not instantaneously activated and initially provides little resistance to extension. The Ca2+-containing EGTA buffer

R. L. MOSS

188 A

c

0-6

CA

pCa 5 49

w

0-2

1-3 cc

1.5 1-7 1 9 Average sarcomere length (Mm)

2-3

2-1

1-0 B

0-8

w

0-6 pCa

6 09

z 0-4

02

0

1-3

1-5 1 7 1.9 Average sarcomere length (Mm)

2-1

2-3

Fig. 8. Plots of steady developed tension as a function of sarcomere length, at two different calcium concentrations, both at 5 0C. The data in A was obtained at pCa 5 49; that in B, at pCa 6*09. In both parts, the filled circles are data points for which the average sarcomere length was determined from end-to-end photomicrographs such as that of PI. 1. The error bars represent + 1 standard deviation of the sarcomere length determination. The open circles are data points for which sarcomere length could not be measured directly over the whole of the segment. These points are plotted vs. an estimated sarcomere length, as explained in the text. Tension is normalized in the manner explained in the legend of Fig. 6. The continuous lines are from Gordon et al. (1966b). All data points were obtained from chemically skinned segments.

LENGTH-TENSION RELATIONS OF SKINNED MUSCLE 189 diffuses into these regions, which can then resist further lengthening. These somewhat longer sarcomere lengths at the ends will tend to increase both the over-all average sarcomere length and the calculated standard deviation. Chemically skinned segments were next activated in solutions of pCa 6-09 to test whether the non-uniformity observed above in fact resulted from the high Ca2+ concentration which was used. This data are plotted in Fig. 8B. It can be seen here that the standard deviations of the sarcomere length measurements are much less than those obtained in solutions of pCa 5*49. Also, sarcomere length measurements were possible at much lower over-all segment lengths. Sarcomere length in relaxed 8egment8 after activation at short length. A living frog muscle fibre which is stimulated when slack, becomes taut during contraction and again becomes slack during relaxation. It is not possible, without special techniques (Gonzales-Serratos, 1971), to obtain sarcomere lengths much below 20 /um in relaxed fibres before the fibres become slack. However, in the present study, when skinned segments were made to contract to sarcomere lengths between about 1-75 and 2-0 Mom, there was usually very little change in sarcomere length upon relaxation. In other instances, when segments were allowed to shorten to sarcomere lengths less than about 1-75 /,m the segment re-extended upon relaxation and became slack. The sarcomere lengths measured in the slackened segments ranged from 1-75 to 1-84 /&m. If this procedure was repeated a number of times the sarcomere length attained following relaxation gradually decreased. In several cases, following many such cycles, sarcomere lengths as low as 1-6 to 1-7 Elm were measured in the relaxed segments. The basis for the re-extension of shortened sarcomeres during relaxation in living fibres is not known. Whatever the cause, the forces involved must be relatively weak, since there was no consistent tendency for the length-tension data from the skinned fibres to lie above the data from the living fibres in the sarcomere length range 1-7-2-0 Em. DISCUSSION

The skinned fibre preparation seems an ideal preparation to use in an investigation of possible effects of length-dependent activation on tension development, since the concentration of calcium in the vicinity of the myofibrils can be maintained constant while sarcomere length is varied. Early in the present investigation, it was apparent that striation patterns in skinned fibre segments were poorly maintained during contractions in solutions containing high, or even moderate, levels of free calcium. A clamping device for grasping the ends of the fibre segments was developed, resulting in a substantial improvement in striation pattern uniformity. Still, in many cases the striations of a segment simply disappeared either totally or in part during activation. When this occurred it was not possible to relate the measured tension, which could be very large, to a meaningful sarcomere length. The main purpose of this investigation was to relate tension to sarcomere length, and for this reason preparations which were observed to have severe structural irregularities during activation were excluded from further experimentation. The primary results of this study indicate that the ascending limb of the sarcomere length-tension relation, obtained in solutions containing constant and relatively high concentrations of free calcium, reproduce the major features of the lengthtension relation described by Gordon et al. (1966b). Between sarcomere lengths of about 2*2 and 1-7 ,um the results reported here agree well with those of Schoenberg & Podolsky (1972), obtained using mechanically skinned muscle fibre segments. However, they reported a substantial elevation of the steep part of the ascending

R. L. MOSS 190 limb relative to the results obtained by Gordon et al. (1966b). The main difference between the present study and that of Schoenberg & Podolsky (1972) is the method of obtaining values for the sarcomere lengths. The light microscopy techniques used in the present study allowed direct measurement of sarcomere length over most or all of the segment, whereas their sarcomere length values were obtained by a laser diffraction technique. According to these authors this method did not work well at short lengths and required that sarcomere length be estimated on the basis of over-all segment length changes. Thus, for most of their measurements at sarcomere lengths less than about 2-0 Aum, direct measurements of striation spacings were not made. One possible explanation for the findings of Schoenberg & Podolsky (1972) is that parts of their fibres shortened at the expense of other parts. In this case, the sarcomere length estimated on the basis of over-all segment length would be misleading. Schoenberg & Podolsky frequently checked the tension generating capabilities of their segments at a reference length. This was done in the present study as well, but in addition the segments were viewed through a microscope. Many segments which developed substantial tensions at the reference length were found to have regions in which striations were non-uniform or had irreversibly disappeared. The results of Taylor & Rudel (1970) and Rudel & Taylor (1971) cannot be simply explained in terms of the present findings. They reported that application of caffeine, in Ringer solution at 20 0C, to tetanically contracting frog muscle fibres at sarcomere lengths less than about 2-5 /sm, increased the amount of steady tension which was developed. This effect became relatively larger as sarcomere length was progressively reduced below about 1-6 tzm. Caffeine was also found to eliminate the wavy myofibrils which. otherwise appeared in the cores of fibres during tetanic stimulation at these short sarcomere lengths, which led to the conclusion that caffeine sustained the activation of the central myofibrils. Since caffeine potentiates the release of calcium from the sarcoplasmic reticulum (Weber, 1968), Taylor & Rudel (1970) suggested that the amount of calcium which is released during tetanic stimulation of living muscle at short sarcomere lengths is insufficient to produce full activation of contraction.In the present study, which was done at 5 0C, no evidence was found of wavy myofibrils in either the skinned or living fibres during steady contractions at sarcomere lengths less than 1-6/jm. This dissimilarity in fibre behaviour may be a result of the different muscles and frog species which were used or possibly a difference in experimental conditions, such as temperature. To test whether caffeine has a direct effect on the myofilaments to enhance tension development, several tension measurements were done on chemically skinned segments at sarcomere lengths of about 1-50 and 2-15 #sm, in solutions of pCa 5*49 containing 0, or 10 mM-caffeine. No discernible differences were observed in the amounts of developed tension at either sarcomere length when the concentration of caffeine was varied. The rate of rise of tension at both sarcomere lengths was found to increase as the caffeine was increased, possibly indicating the presence of a small amount of intact sarcoplasmic reticulum, which is largely destroyed by chemically skinning (Moss et al. 1976). 1

Blinks, Rudel & Taylor (1978) have measured the light responses of fibres which were injected with the photoprotein aequorin, which can be used to determine the level of free calcium, and were then made to contract tetanically. They found that the peak light responses during contraction declined only gradually as sarcomere length

191 LENGTH-TENSION RELATIONS OF SKINNED MUSCLE was reduced from 2-4 to 1-4 #m. Thus, over most of the steep part of the ascending limb, the free calcium ion concentration during tetanic stimulation appears to vary only a small amount. It has already been pointed out that changes in the amount of calcium which is released from the sarcoplasmic reticulum may produce sizeable changes in the free calcium ion concentration while at the same time having only a slight effect on the amount of calcium which is bound to the regulatory proteins (Homsher & Kean, 1978). The results of the present study indicate that the shape of the ascending limb obtained from skinned fibres in solutions having fixed calcium concentrations, is similar to the ascending limb obtained from living fibres during tetanic contractions. This finding is not consistent with the view that a sarcomere length-dependent variation in the level of activator calcium plays a major role in determining the shape of the ascending limb in living fibres. The most probable explanation for the basis of the ascending limb involves mechanical and structural factors, as suggested by Gordon et al. (1966b), though the results here make it seem unlikely that hydrostatic pressure contributes a significant force opposing contraction at short lengths. I am grateful to Dr F. J. Julian for his support and assistance. This work was supported by the following grants: an N.I.H. research grant, HL-16606, from the National Heart, Lung and Blood Institute, and grants from the American Heart Association, No. 77-616, and the Muscular Dystrophy Association. REFERENCES

BLINKS, J. R., RUDEL, R. & TAYLOR, S. R. (1978). Calcium transients in isolated amphibian skeletal muscle fibres: Detection with aequorin, J. Physiol. 277, 291-323. CAMBRIDGE, G. W. & HAINEs, J. (1959). A new versatile transducer system. J. Physiol. 149, 23P. GODT, R. E. (1974). Calcium-activated tension of skinned muscle fibres of the frog. J. gen. Physiol. 63, 722-739. GONZALES-SERRATOS, H. (1971). Inward spread of activation in vertebrate muscle fibres. J. Physiol. 212, 777-799. GORDON, A. M., HuxLEY, A. F. & JULIAN, F. J. (1966a). Tension development in highly stretched vertebrate muscle fibres. J. Physiol. 184, 143-169. GORDON, A. M., HuxLEY, A. F. & JuLIAkN, F. J. (1966b). The variation in isometric tension with sarcomere length in vertebrate muscle fibres. J. Phygiol. 184, 170-192. HOMSHER, E. & KEAN, C. J. (1978). Skeletal muscle energetic and metabolism. A. Rev. Phy8iol. 40, 93-131. Huxiy, A. F. & PEACHEY, L. D. (1961). The maximum length for contraction in vertebrate striated muscle. J. Physiol. 156, 150-165. JULIAN, F. J. (1971). The effect of calcium on the force-velocity relation of briefly glycerinated frog muscle fibres. J. Phy8iol. 218, 117-145. JuLIAN, F. J. & SOLLINs, M. R. (1973). Regulation of force and speed of shortening in muscle contraction. Cold Spring Harb. Symp. quant. Biol. 37, 635-646. JUIiAN, F. J. & SOLLINS, M. R. (1975). Variation of muscle stiffness with force at increasing speeds of shortening. J. gen. Phy8iol. 66, 287-302. JuLIAN, F. J., SoLLINs, M. R. & Moss, R. L. (1978). Sarcomere length non-uniformity in relation to tetanic responses of stretched skeletal muscle fibres. Proc. R. Soc. Lond. B. 200, 109-116.

MOIsEsCu, D. G. (1976). Kinetics of reaction in Ca-activated skinned muscle fibres. Nature, Lond. 262, 610-613. Moss, R. L. & JuLIAN, F. J. (1978). Sarcomere length-tension relations in living and calcium activated muscle fibers. Biophy8. J. 21, 63a. Moss, R. L., SoLLiNs, M. R. & JuLIAN, F. J. (1976). Calcium activation produces a characteristic response to stretch in both skeletal and cardiac muscle. Nature. Lond. 260, 619-621. Moss, R. L., SoLLINs, M. R. & JurmwN, F. J. (1977). Sarcomere length-tension relations in

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tetanically stimulated and calcium activated frog skeletal muscle fibers below optimum length. Biophy8. J. 17, 174a. NATORI, R. (1954). The property and contraction process of isolated myofibrils. Jikei med. J. 1, 119-126. RUBsEy, R. W. & STREET, S. F. (1940). The isometric length-tension diagram of isolated skeletal muscle fibers of the frog. J. ceU. comp. Phyeiol. 15, 11-34. RUDEL, R. & TAYLOR, S. R. (1971). Striated muscle fibers: facilitation of contraction at short lengths by caffeine. Science, N.Y. 172, 387-388. SCHOENBERG, M. & PODOLsKY, R. J. (1972). Length-force relation of calcium activated muscle fibers. Science. N.Y. 172, 52-54. TAYLOR, S. R. & RUDEL, R. (1970). Striated muscle fibers: inactivation of contraction induced by shortening. Science, N.Y. 167, 882-884. WEBEiR, A. (1968). The mechanism of action of caffeine on sarcoplasmic reticulum. J. gen. Physiol. 52, 760-772. EXPLANATION OF PLATES

PLATE 1 Low power light photomicrograph of fibre segment of 28 February, 1978, indicating the method of sarcomere length determination. In A the segment is shown while in relaxing solution. The length of the segment is 0-89 mm and the width is about 120 jsm. Small portions of the connectors are visible at each segment end. The original film negative of this micrograph was mounted in a photographic enlarger, and the image was projected on smooth, white paper. A tracing was made of this image, and three rows of striations were marked off in intervals of fifty striations each, as shown in B. An average sarcomere length was then measured for each interval, indicated by the numbers above the intervals. Sarcomere length was calculated from the entire segment as the average of the twenty-one individual determinations. The segment was chemically skinned. The bar represents 100 jsm.

PLATE 2 of 3 October 1977. In A, the segment was in relaxing of the segment Light photomicrographs solution. The length of the segment was 0-62 mm, and the width was about 71 am. The average sarcomere length was measured to be 2.20 + 0.06 (S.D.) jam. In B, the segment was photographed while the segment was fully activated in a solution of pCa 5-49. The average sarcomere length was in this instance 2-19 ± 0-12 (S.D.) jAm. The segment was chemically skinned.

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