719
Journal of Physiology (1991), 441, pp. 719-732 With 6 figu1res Printed in Great Britain
TENSION AS A FUNCTION OF SARCOMERE LENGTH AND VELOCITY OF SHORTENING IN SINGLE SKELETAL MUSCLE FIBRES OF THE FROG
BY DAVID L. MORGAN*, DENNIS R. CLAFLINt AND FRED J. JULIAN From the Department of Anesthesia Research Laboratories, Harvard Medical School, Brigham & Women's Hospital, Boston, MA 02115 USA
(Received 19 November 1990) SUMMARY
1. Simple measurements of muscle tension at fixed fibre or segment length produce a range of length-tension relationships, depending primarily on the duration of the interval between stimulation onset and tension measurement, in contradiction with the simple predictions of current models. This has been explained by non-uniformity in sarcomere lengths, leading to internal motion and, in turn, to increasing tension because the force-velocity relationship has a much greater slope for slow lengthening than for slow shortening. 2. Previous attempts to reduce the effect of internal motion have been focused on decreasing the initial extent of non-uniformity and measuring tension early in a contraction, when non-uniformities are at a minimum. An alternative approach that has not been attempted previously is to reduce the non-linearity of the force-velocity relationship by avoiding the discontinuity in slope at zero velocity. This is accomplished by imposing overall fibre shortening at velocities sufficient to ensure that all sarcomeres are shortening. 3. When the tension maintained during shortening was measured and plotted against sarcomere length for each release velocity used, linear length-tension relationships resulted that extrapolated to a common sarcomere length intercept. This was true whether the release was applied early in the tetanus or near the end of the 'creep phase' of tension rise. These observations were duplicated by computer simulation using a multisarcomere model of a muscle fibre. 4. These results provide strong support for the view that cross-bridges function as independent force generators and for the explanation of the creep phase of fibre or segment isometric tension as being due to internal motion. The results also imply that the force-velocity relationship scales with sarcomere length without changing shape. 5. Using this novel method for obtaining length-tension relationships, the sarcomere length at which active tension fell to zero was found, by extrapolation, to *
Present address: Department of Electrical and Computer Systems Engineering, Monash
University, Clayton, Victoria 3168, Australia. t To whom all correspondence should be addressed. MS 8939
720
D. L. MORGAN, D. R. CLAFLIN AND F. J. JULIAN
be 3 65,um in semitendinosus fibres and 3 53,um in tibialis anterior fibres from the frog (Rana temporaria). INTRODUCTION
One common feature of cross-bridge theories of the type first proposed by Huxley (1957) is that cross-bridges function as independent force generators. This leads to the prediction that the tension-generating capacity of skeletal muscle is directly proportional to the overlap of thick and thin filaments. However, it has been clear for many years that the tension in a frog skeletal muscle fibre, measured late in a tetanic contraction either at fixed fibre length or fixed segment length, is not proportional to filament overlap (Ramsey & Street, 1940; Huxley & Peachey, 1961; Julian & Morgan, 1979; Edman & Regiani, 1984; Altringham & Bottinelli, 1985; Granzier & Pollack, 1990). This discrepancy is often attributed to tension 'creep', the slow phase of tension rise that follows the fast phase at long sarcomere lengths. Controlling the length of a segment slows the rate of rise of the creep phase, but does not abolish it (Julian & Morgan, 1979). It does, however, allow more accurate measurements of the rapid phase of tension rise, which are consistent with the view that cross-bridges function as independent force generators (Gordon, Huxley & Julian, 1966). It has long been argued that tension creep is due to internal motion caused by sarcomere non-uniformities (Huxley & Peachey, 1961; Gordon et al. 1966; Julian, Sollins & Moss, 1978; Edman & Regiani, 1984; Morgan, 1985). Consider the force-velocity characteristics of two sarcomeres shown as plots of P/PO against V/ V. in Fig. IA, where P is the load, PO is isometric tetanic tension at optimum length, V is shortening velocity and V. is unloaded shortening velocity. Because the two sarcomeres are not at the same length and not at the optimum length for tension generation, they have isometric capabilities (the intersections with the zero-velocity line) of 0-6 and 0-8 of PO. Note that the non-uniformities may well be between halfsarcomeres rather than sarcomeres, and the use of the term sarcomeres is only for convenience. The curves shown are hyperbolae with a/PO of 0-25 (Hill, 1938), and with a 6:1 slope change at zero velocity (Katz, 1939). If these two sarcomeres are connected in series and the overall length is held constant ('isometric' contraction), the sarcomere tensions must be equal and the sum of the sarcomere velocities must be zero. That is, the two sarcomere velocities must be of equal magnitude and opposite sign. The tension at which these conditions are satisfied is P/P. = 0-770 (horizontal dotted line, filled circles), higher than the average of the two isometric capabilities, which is P/PO = 0 7 (horizontal dashed line). If the descending limb of the length-tension relationship is linear, then some time later in the contraction the isometric capabilities would be P/PO = 0-4 and 1D0 (Fig. 1B), due to the equal and opposite velocities. At this point the tension required to produce zero overall velocity is higher still at P/PO = 0 904 (horizontal dotted line, filled circles), while the average isometric capability remains P/PO = 0 7 (horizontal dashed line). That is, the 'isometric' tension of the series combination of the two sarcomeres is always higher than the average of the isometric tensions of the sarcomeres taken individually, and increases with increasing non-uniformity as the contraction proceeds.
MUSCLE LENGTH-TENSION DURING SHORTENING A
721
B
A -0 1.0-
1.01F 0
0
a)
20 0.5 -
Sarcomere 1
5.
U-
~~~~~~~1
\
\
0
U-
I2 0.0
0.1
0.2
0-0
0.3
:~~~~ 0.0
0-1
0-2
0.3
0.4
Velocity (V/Vu)
Velocity (VIVu)
D
C
&10
100.0
0
0L
1
0
O"
0.5-
o
Q.5
0
7
,, .. 0-0
0-1
0-2
0-3
0-4
Velocity (VIVu) VelocitY (oV/Vu) 1. Each a of Fig. panel shows segment the force-velocity curve of each of two individual sarcomeres. P is load, P. is isometric tetanic tension at optimum length, V is shortening velocity, and Vu is unloaded shortening velocity. In A and C, the sarcomeres have isometric capabilities of 0-6 and 0-8 of P.. In B and D the isometric capabilities are 0-4 and 1-0 PO, representing the same two sarcomeres later in a contraction. In A and B the overall or average velocity is zero, while in C and D the average sarcomere velocity is 20 % of Vu. In each case the horizontal dotted line and filled circles represent the tension that produces the required average velocity. When the average velocity is zero (A and B), the tension is significantly different from the average of the sarcomere isometric tensions (horizontal dashed lines), and the difference increases with the degree of non-uniformity. This difference is indicated by the vertical arrows. When the average velocity is non-zero (C and D), the tension is much closer to the average of the tensions of the two sarcomeres if each shortened at the average velocity (horizontal dashed lines), and relatively independent of the degree of non-uniformity. Note that the difference between the two tensions is not distinguishable early in the contraction (C) and is still small late in the contraction (D, arrows).
Attempts to overcome this problem have traditionally concentrated on reducing the degree of initial sarcomere non-uniformity in the fibre segment being held at constant length. However, since the discontinuity in the slope of the force-velocity
722
D. L. MORGAIN, D. R. CLAFLIN ANVD F J. JULIAN
relationship at zero velocity is equally crucial to the explanation of creep, elimination of the discontinuity also should result in improved measurements of the length-tension relationship. This can be accomplished by applying an overall fibre shortening velocity sufficient to ensure that all sarcomeres are shortening. Consider again the sarcomeres in Fig. 1, now shortening at a velocity of V/Vu = 0-2 (Fig. 1 C). At this velocity, the tension in a sarcomere with an isometric capability of P/P. = 0-7, the average sarcomere, is P/PO = 0 31 1 (horizontal dashed line). If two sarcomeres are connected in series, their average velocity must equal the imposed velocity. The tension required to satisfy this condition for two sarcomeres with isometric capabilities of 0 6 and 0-8 of P. and a velocity of V/Vu = 0-2 is P/PO = 0 309 (filled circles). If the isometric capabilities are 0 4 and I 0 PO (Fig. ID), then the required tension is P/PO = 0 290 (horizontal dotted line, filled circles). This shows, for a twosarcomere model, that the degree of sarcomere non-uniformity affects the tension during shortening much less than it affects the tension when average sarcomere velocity is zero. We report here experiments that show that this is also true for intact muscle fibres and for models with more than two sarcomeres. For any particular velocity of shortening, tension varies with length as expected if cross-bridges act as independent force generators in the overlap zone, provided that overall or average velocities near zero are avoided. The sarcomere length at which tension generation falls to zero is predicted to be the sarcomere length at which thick and thin filaments no longer overlap. Bagni, Cecchi, Colomo & Tesi (1988) have suggested that this length is different for fibres from the tibialis anterior muscle than for the semitendinosus fibres used by Gordon et al. (1966). This difference was confirmed by performing the experiments on both types of fibres. Brief reports of these results have been presented to the American Biophysical Society (Claflin, Morgan & Julian, 1990, 1991). METHODS
Experiments were performed on single living fibres from the frog (Rana temporaria) using the preparative methods and equipment previously described (Julian & Morgan, 1979; Julian, Rome, Stephenson & Striz, 1986; Claflin, Morgan & Julian, 1989). Frogs were killed by decapitation followed by double pithing immediately upon removal from cold (4 °C) storage. Fibres were then isolated from either the dorsal head of the semitendinosus muscle or the ventral head of the tibialis anterior muscle. Briefly, fibres were mounted in a chamber by attaching one tendon to a tension transducer and the other to a servomotor. The temperature of the Ringer solution in the chamber was maintained at 2 5 + 0-2 °C throughout all experiments. Average sarcomere length was determined from photographs taken at 1 mm intervals along the tautly stretched fibre. The fibre length was then divided by the average sarcomere length to determine the number of sarcomeres in series. Subsequen-t sarcomere lengths were set by stretching the fibre to lengths determined by multiplying the desired sarcomere length by the number of sarcomeres in series. Previous experience with the mounting technique used in these experiments has shown that mounting compliance is negligible, no more than a few nanometres per sarcomere (Julian & Morgan, 1981). The basic experiment consisted of applying a constant-velocity (ramp) shortening to an activated fibre, and measuring the tension at the time it first became nearly constant. Several typical records are shown in Fig. 2. The tension levels that were recorded are indicated by the horizontal dashed lines. It should be emphasized that the results did not depend critically on the method used to determine the tension during shortening. Nevertheless, a well-defined, observerindependent method was devised. The point to be read was defined as the point where the rate of
MUSCLE LENGTH-TENSION, DURIVG SHORTENING
723
fall of active tension declined to 5% of its maximum value, excluding the rapid transient that immediately followed release. The average sarcomere length associated with each tension value was calculated by dividing the length of the fibre at the time the tension was read by the number of sarcomeres in series. 2.8 gm Length
15\
Tension
2 7 ,um
.-..
Fig. 2. Typical experimental records. Movements of the servomotor, calibrated as average sarcomere length, are shown superimposed in the upper traces. Superimposed tension responses are shown below. Releases were made at constant velocities of 50, 30, 20 and 10 % of Vu from an average sarcomere length of 2-8 ,um to an average sarcomere length of 2-7 ,um. The horizontal dashed lines in the lower panel indicate the level of tension recorded for each release velocity. The records shown are from a semitendinosus fibre released 1500 ms after the onset of stimulation. Duration of stimulation was 1700 ms.
For the twelve fibres (six of each type) reported here, ramps of magnitude sufficient to decrease average sarcomere length by 01 ,um were initiated from sarcomere lengths of (in /um) 2-05, 2-10, 2-15, 2-20, 2-30, 2-40, 2-60, 2-80, 3-00, 3-20 and, finally, 2-60 again as a control. From each length, ramps with speeds of 50, 30, 20 and 10% of Vu were applied 500 ms after the onset of a 700 ms tetanic stimulation (35 pulses/s). From most lengths, ramps of the same speeds were also applied 1500 ms after the onset of a 1700 ms stimulation in a separate contraction. Early (500 ms) and late (1500 ms) releases were alternated. Experiments proceeded in order from shortest sarcomere length to longest, with ramps of each speed applied before increasing length. Tetanic stimulations were separated by 180 s recovery periods. V. was determined at optimal length for tension generation as the slowest ramp speed required to reduce the tension maintained during shortening to zero. Maximum tension-generating capacity (PO) was defined as the tension obtained 500 ms after initiation of stimulation at a sarcomere length of 2-05 ,um for the tibialis anterior fibres and 2-15 ,tm for the semitendinosus fibres. At the end of an experiment, the P. values were 94 + 2 and 88 + 3 % of the original levels for the semitendinosus and tibialis fibres, respectively (means + S.E.M., n = 6). To compensate for this small decline, PO was measured before each length change (every nine, or fewer, contractions) and all tension results are reported as a percentage of a PO value obtained by linear interpolation between two measured values. The zero-velocity tension was taken from the records as the tension just before the ramp began, averaged over all the releases from a particular length. Corrections were made for passive tension by subtracting from each active tension record a tension record obtained by applying the same ramp to the fibre while not stimulated. For average sarcomere lengths shorter than 3 0 ,um for semitendinosus fibres and 2-8 ,um for tibialis fibres, this 'dynamic passive tension' was negligible and no adjustment was necessary. At longer lengths, the dynamic passive tension made a significant contribution to total tension. From a given sarcomere length, all contractions were initiated from a constant level of passive tension. This was achieved while maintaining the 180 s recovery period by varying the time at which the fibre was restretched following the previous ramp release (see Claflin et al. 1989).
724
7D. L. MORGANS, D. R. CLAFLIN AND F. J. JULIAN RESULTS
Typical records of a complete series of tension responses to releases from one sarcomere length are shown in Fig. 3. The similarity between tension during a release applied early in a contraction (horizontal dashed lines) and tension during a release
-t/
10%
|
Journal of Physiology (1991), 441, pp. 719-732 With 6 figu1res Printed in Great Britain
TENSION AS A FUNCTION OF SARCOMERE LENGTH AND VELOCITY OF SHORTENING IN SINGLE SKELETAL MUSCLE FIBRES OF THE FROG
BY DAVID L. MORGAN*, DENNIS R. CLAFLINt AND FRED J. JULIAN From the Department of Anesthesia Research Laboratories, Harvard Medical School, Brigham & Women's Hospital, Boston, MA 02115 USA
(Received 19 November 1990) SUMMARY
1. Simple measurements of muscle tension at fixed fibre or segment length produce a range of length-tension relationships, depending primarily on the duration of the interval between stimulation onset and tension measurement, in contradiction with the simple predictions of current models. This has been explained by non-uniformity in sarcomere lengths, leading to internal motion and, in turn, to increasing tension because the force-velocity relationship has a much greater slope for slow lengthening than for slow shortening. 2. Previous attempts to reduce the effect of internal motion have been focused on decreasing the initial extent of non-uniformity and measuring tension early in a contraction, when non-uniformities are at a minimum. An alternative approach that has not been attempted previously is to reduce the non-linearity of the force-velocity relationship by avoiding the discontinuity in slope at zero velocity. This is accomplished by imposing overall fibre shortening at velocities sufficient to ensure that all sarcomeres are shortening. 3. When the tension maintained during shortening was measured and plotted against sarcomere length for each release velocity used, linear length-tension relationships resulted that extrapolated to a common sarcomere length intercept. This was true whether the release was applied early in the tetanus or near the end of the 'creep phase' of tension rise. These observations were duplicated by computer simulation using a multisarcomere model of a muscle fibre. 4. These results provide strong support for the view that cross-bridges function as independent force generators and for the explanation of the creep phase of fibre or segment isometric tension as being due to internal motion. The results also imply that the force-velocity relationship scales with sarcomere length without changing shape. 5. Using this novel method for obtaining length-tension relationships, the sarcomere length at which active tension fell to zero was found, by extrapolation, to *
Present address: Department of Electrical and Computer Systems Engineering, Monash
University, Clayton, Victoria 3168, Australia. t To whom all correspondence should be addressed. MS 8939
720
D. L. MORGAN, D. R. CLAFLIN AND F. J. JULIAN
be 3 65,um in semitendinosus fibres and 3 53,um in tibialis anterior fibres from the frog (Rana temporaria). INTRODUCTION
One common feature of cross-bridge theories of the type first proposed by Huxley (1957) is that cross-bridges function as independent force generators. This leads to the prediction that the tension-generating capacity of skeletal muscle is directly proportional to the overlap of thick and thin filaments. However, it has been clear for many years that the tension in a frog skeletal muscle fibre, measured late in a tetanic contraction either at fixed fibre length or fixed segment length, is not proportional to filament overlap (Ramsey & Street, 1940; Huxley & Peachey, 1961; Julian & Morgan, 1979; Edman & Regiani, 1984; Altringham & Bottinelli, 1985; Granzier & Pollack, 1990). This discrepancy is often attributed to tension 'creep', the slow phase of tension rise that follows the fast phase at long sarcomere lengths. Controlling the length of a segment slows the rate of rise of the creep phase, but does not abolish it (Julian & Morgan, 1979). It does, however, allow more accurate measurements of the rapid phase of tension rise, which are consistent with the view that cross-bridges function as independent force generators (Gordon, Huxley & Julian, 1966). It has long been argued that tension creep is due to internal motion caused by sarcomere non-uniformities (Huxley & Peachey, 1961; Gordon et al. 1966; Julian, Sollins & Moss, 1978; Edman & Regiani, 1984; Morgan, 1985). Consider the force-velocity characteristics of two sarcomeres shown as plots of P/PO against V/ V. in Fig. IA, where P is the load, PO is isometric tetanic tension at optimum length, V is shortening velocity and V. is unloaded shortening velocity. Because the two sarcomeres are not at the same length and not at the optimum length for tension generation, they have isometric capabilities (the intersections with the zero-velocity line) of 0-6 and 0-8 of PO. Note that the non-uniformities may well be between halfsarcomeres rather than sarcomeres, and the use of the term sarcomeres is only for convenience. The curves shown are hyperbolae with a/PO of 0-25 (Hill, 1938), and with a 6:1 slope change at zero velocity (Katz, 1939). If these two sarcomeres are connected in series and the overall length is held constant ('isometric' contraction), the sarcomere tensions must be equal and the sum of the sarcomere velocities must be zero. That is, the two sarcomere velocities must be of equal magnitude and opposite sign. The tension at which these conditions are satisfied is P/P. = 0-770 (horizontal dotted line, filled circles), higher than the average of the two isometric capabilities, which is P/PO = 0 7 (horizontal dashed line). If the descending limb of the length-tension relationship is linear, then some time later in the contraction the isometric capabilities would be P/PO = 0-4 and 1D0 (Fig. 1B), due to the equal and opposite velocities. At this point the tension required to produce zero overall velocity is higher still at P/PO = 0 904 (horizontal dotted line, filled circles), while the average isometric capability remains P/PO = 0 7 (horizontal dashed line). That is, the 'isometric' tension of the series combination of the two sarcomeres is always higher than the average of the isometric tensions of the sarcomeres taken individually, and increases with increasing non-uniformity as the contraction proceeds.
MUSCLE LENGTH-TENSION DURING SHORTENING A
721
B
A -0 1.0-
1.01F 0
0
a)
20 0.5 -
Sarcomere 1
5.
U-
~~~~~~~1
\
\
0
U-
I2 0.0
0.1
0.2
0-0
0.3
:~~~~ 0.0
0-1
0-2
0.3
0.4
Velocity (V/Vu)
Velocity (VIVu)
D
C
&10
100.0
0
0L
1
0
O"
0.5-
o
Q.5
0
7
,, .. 0-0
0-1
0-2
0-3
0-4
Velocity (VIVu) VelocitY (oV/Vu) 1. Each a of Fig. panel shows segment the force-velocity curve of each of two individual sarcomeres. P is load, P. is isometric tetanic tension at optimum length, V is shortening velocity, and Vu is unloaded shortening velocity. In A and C, the sarcomeres have isometric capabilities of 0-6 and 0-8 of P.. In B and D the isometric capabilities are 0-4 and 1-0 PO, representing the same two sarcomeres later in a contraction. In A and B the overall or average velocity is zero, while in C and D the average sarcomere velocity is 20 % of Vu. In each case the horizontal dotted line and filled circles represent the tension that produces the required average velocity. When the average velocity is zero (A and B), the tension is significantly different from the average of the sarcomere isometric tensions (horizontal dashed lines), and the difference increases with the degree of non-uniformity. This difference is indicated by the vertical arrows. When the average velocity is non-zero (C and D), the tension is much closer to the average of the tensions of the two sarcomeres if each shortened at the average velocity (horizontal dashed lines), and relatively independent of the degree of non-uniformity. Note that the difference between the two tensions is not distinguishable early in the contraction (C) and is still small late in the contraction (D, arrows).
Attempts to overcome this problem have traditionally concentrated on reducing the degree of initial sarcomere non-uniformity in the fibre segment being held at constant length. However, since the discontinuity in the slope of the force-velocity
722
D. L. MORGAIN, D. R. CLAFLIN ANVD F J. JULIAN
relationship at zero velocity is equally crucial to the explanation of creep, elimination of the discontinuity also should result in improved measurements of the length-tension relationship. This can be accomplished by applying an overall fibre shortening velocity sufficient to ensure that all sarcomeres are shortening. Consider again the sarcomeres in Fig. 1, now shortening at a velocity of V/Vu = 0-2 (Fig. 1 C). At this velocity, the tension in a sarcomere with an isometric capability of P/P. = 0-7, the average sarcomere, is P/PO = 0 31 1 (horizontal dashed line). If two sarcomeres are connected in series, their average velocity must equal the imposed velocity. The tension required to satisfy this condition for two sarcomeres with isometric capabilities of 0 6 and 0-8 of P. and a velocity of V/Vu = 0-2 is P/PO = 0 309 (filled circles). If the isometric capabilities are 0 4 and I 0 PO (Fig. ID), then the required tension is P/PO = 0 290 (horizontal dotted line, filled circles). This shows, for a twosarcomere model, that the degree of sarcomere non-uniformity affects the tension during shortening much less than it affects the tension when average sarcomere velocity is zero. We report here experiments that show that this is also true for intact muscle fibres and for models with more than two sarcomeres. For any particular velocity of shortening, tension varies with length as expected if cross-bridges act as independent force generators in the overlap zone, provided that overall or average velocities near zero are avoided. The sarcomere length at which tension generation falls to zero is predicted to be the sarcomere length at which thick and thin filaments no longer overlap. Bagni, Cecchi, Colomo & Tesi (1988) have suggested that this length is different for fibres from the tibialis anterior muscle than for the semitendinosus fibres used by Gordon et al. (1966). This difference was confirmed by performing the experiments on both types of fibres. Brief reports of these results have been presented to the American Biophysical Society (Claflin, Morgan & Julian, 1990, 1991). METHODS
Experiments were performed on single living fibres from the frog (Rana temporaria) using the preparative methods and equipment previously described (Julian & Morgan, 1979; Julian, Rome, Stephenson & Striz, 1986; Claflin, Morgan & Julian, 1989). Frogs were killed by decapitation followed by double pithing immediately upon removal from cold (4 °C) storage. Fibres were then isolated from either the dorsal head of the semitendinosus muscle or the ventral head of the tibialis anterior muscle. Briefly, fibres were mounted in a chamber by attaching one tendon to a tension transducer and the other to a servomotor. The temperature of the Ringer solution in the chamber was maintained at 2 5 + 0-2 °C throughout all experiments. Average sarcomere length was determined from photographs taken at 1 mm intervals along the tautly stretched fibre. The fibre length was then divided by the average sarcomere length to determine the number of sarcomeres in series. Subsequen-t sarcomere lengths were set by stretching the fibre to lengths determined by multiplying the desired sarcomere length by the number of sarcomeres in series. Previous experience with the mounting technique used in these experiments has shown that mounting compliance is negligible, no more than a few nanometres per sarcomere (Julian & Morgan, 1981). The basic experiment consisted of applying a constant-velocity (ramp) shortening to an activated fibre, and measuring the tension at the time it first became nearly constant. Several typical records are shown in Fig. 2. The tension levels that were recorded are indicated by the horizontal dashed lines. It should be emphasized that the results did not depend critically on the method used to determine the tension during shortening. Nevertheless, a well-defined, observerindependent method was devised. The point to be read was defined as the point where the rate of
MUSCLE LENGTH-TENSION, DURIVG SHORTENING
723
fall of active tension declined to 5% of its maximum value, excluding the rapid transient that immediately followed release. The average sarcomere length associated with each tension value was calculated by dividing the length of the fibre at the time the tension was read by the number of sarcomeres in series. 2.8 gm Length
15\
Tension
2 7 ,um
.-..
Fig. 2. Typical experimental records. Movements of the servomotor, calibrated as average sarcomere length, are shown superimposed in the upper traces. Superimposed tension responses are shown below. Releases were made at constant velocities of 50, 30, 20 and 10 % of Vu from an average sarcomere length of 2-8 ,um to an average sarcomere length of 2-7 ,um. The horizontal dashed lines in the lower panel indicate the level of tension recorded for each release velocity. The records shown are from a semitendinosus fibre released 1500 ms after the onset of stimulation. Duration of stimulation was 1700 ms.
For the twelve fibres (six of each type) reported here, ramps of magnitude sufficient to decrease average sarcomere length by 01 ,um were initiated from sarcomere lengths of (in /um) 2-05, 2-10, 2-15, 2-20, 2-30, 2-40, 2-60, 2-80, 3-00, 3-20 and, finally, 2-60 again as a control. From each length, ramps with speeds of 50, 30, 20 and 10% of Vu were applied 500 ms after the onset of a 700 ms tetanic stimulation (35 pulses/s). From most lengths, ramps of the same speeds were also applied 1500 ms after the onset of a 1700 ms stimulation in a separate contraction. Early (500 ms) and late (1500 ms) releases were alternated. Experiments proceeded in order from shortest sarcomere length to longest, with ramps of each speed applied before increasing length. Tetanic stimulations were separated by 180 s recovery periods. V. was determined at optimal length for tension generation as the slowest ramp speed required to reduce the tension maintained during shortening to zero. Maximum tension-generating capacity (PO) was defined as the tension obtained 500 ms after initiation of stimulation at a sarcomere length of 2-05 ,um for the tibialis anterior fibres and 2-15 ,tm for the semitendinosus fibres. At the end of an experiment, the P. values were 94 + 2 and 88 + 3 % of the original levels for the semitendinosus and tibialis fibres, respectively (means + S.E.M., n = 6). To compensate for this small decline, PO was measured before each length change (every nine, or fewer, contractions) and all tension results are reported as a percentage of a PO value obtained by linear interpolation between two measured values. The zero-velocity tension was taken from the records as the tension just before the ramp began, averaged over all the releases from a particular length. Corrections were made for passive tension by subtracting from each active tension record a tension record obtained by applying the same ramp to the fibre while not stimulated. For average sarcomere lengths shorter than 3 0 ,um for semitendinosus fibres and 2-8 ,um for tibialis fibres, this 'dynamic passive tension' was negligible and no adjustment was necessary. At longer lengths, the dynamic passive tension made a significant contribution to total tension. From a given sarcomere length, all contractions were initiated from a constant level of passive tension. This was achieved while maintaining the 180 s recovery period by varying the time at which the fibre was restretched following the previous ramp release (see Claflin et al. 1989).
724
7D. L. MORGANS, D. R. CLAFLIN AND F. J. JULIAN RESULTS
Typical records of a complete series of tension responses to releases from one sarcomere length are shown in Fig. 3. The similarity between tension during a release applied early in a contraction (horizontal dashed lines) and tension during a release
-t/
10%
|
