Journal of Muscle Research and Cell Motility 13, 295-307 (1992)
Contribution of damped passive recoil to the measured shortening velocity of skinned rabbit and sheep muscle fibres C. Y. S E O W *
and L. E. F O R D
Section of Cardiology, Department of Medicine, University of Chicago, Chicago, IL 60637, USA Received 15 February 1991; revised and accepted 20 October 1991
Summary Maximum shortening velocities of skinned fibres from rabbit psoas and sheep extensor digitorum longus muscles were measured by the slack test and by extrapolating force-velocity curves to zero load. Both overall muscle velocity and sarcomere velocity were measured with each method. Maximum sarcomere velocity measured by the slack test was not significantly different from that assessed from the force-velocity curves (p > 0.1). Maximum overall muscle velocity measured from the slack test was significantly (p > 0.001) and substantially (62% rabbit, 83% sheep) greater than maximum sarcomere velocity. The difference is attributed to damped recoil of the series elastic elements contributing to the overall muscle velocity. The extent and time course of this damped recoil in isotonic steps was assessed from comparisons of overall muscle length and sarcomere length records during isotonic steps. When the records were shifted and scaled so that they superposed during the late stages of isotonic shortening, there was a substantial difference between the early parts of the records. This difference was reduced by about half in association with the step and the remaining half declined at a diminishing rate following the step, lasting longer with lower loads. This result is explained by about half of the series elastic element behaving as a viscoelastic element and half being undamped. With steps to the lowest isotonic loads, which averaged 6.7% of isometric force in sheep and 9.5% in rabbit, the total series elastic element recoil (both damped and undamped) averaged 3.4% and 2.7% of fibre segment length, respectively, in sheep and rabbit. The rapid series elastic element recoil at zero load, assessed from the slack test, was approximately 50% higher, indicating a substantial series compliance at low forces. The contribution of an additional, longer lasting, damped series elastic element recoil to the overall muscle velocity can explain the greater maximum velocity that is frequently found with the slack test.
Introduction Maximum shortening velocity of muscle is usually estimated by extrapolating the force-velocity curve to zero load, or by making the muscle go slack from isometric tension and measuring the time taken for force to begin to redevelop (Hill, 1970; Edman, 1979). The latter method, called slack test, has consistently yielded maximum velocities higher than those obtained from force-velocity curves in comparative studies (Edman, 1979; Julian & Moss, 1981; Moss, 1982; L~innergren et al., 1982; Goldman, 1983). As it is not likely that muscle *To whom correspondenceshouldbe addressed at: HospitalBox 249, University of Chicago Hospitals, 5841 South Maryland Avenue, Chicago, IL 60637, USA. 0142-4319 9
1992 Chapman & Hall
possesses distinct maximum velocities under different testing conditions, the discrepancy raises the question of the validity of the methods. In direct comparisons of the two methods, the disparity has ranged from 5% in studies of intact fibres (Edman, 1979) to 50% in skinned fibres (Julian & Moss, 1981). Our previous studies of the force velocity properties of skinned fibres showed that overall muscle velocity was substantially higher than sarcomere velocity at light loads during the early phase of isotonic shortening (Ford et al., 1991, Seow & Ford, 1991b). This difference was explained by damped recoil of the series elastic elements. In the present study we have assessed the effects of this damped recoil on measurements of maximum velocity determined by the two methods mentioned above. Some of these results
296 have been briefly presented in an abstract (Seow & Ford, 1991a).
Materials and methods MUSCLE FIBRE PREPARATION Animals A 55 kg sheep and several rabbits weighing about 2kg were used. Rabbits were first anaesthetized with intramuscular injections of 50 mg ketamine and 20 mg of xylazine, followed by an intracardiac injection of saturated KC1 solution to stop the heart. Muscle tissue from a sheep was obtained from a local abattoir immediately after the animal was slaughtered. Dissection and preparation of muscle bundles from the sheep Strips of extensor digitorum longus (EDL) were removed from the hind limbs of the sheep, tied to glass rods and kept overnight at 0~ in skinning solution (see SOLUTIONS).After the overnight skinning the tissue was soaked for I h in a solution containing 25% (by volume) glycerol and 75% relaxing solution (see SOLUTIONS) at 0 ~ C. Finally, the tissue was transferred to the storage solution containing equal portions of glycerol and relaxing solution. The pH of the storage solution was set to 6.5 at room temperature to provide a pH of 7.0-7.5 at the storage temperature of - 2 0 ~ C.
Dissection and preparation of muscle bundles from the rabbits Strips of psoas muscle were removed from the rabbits, tied to glass rods and kept at 0~ in relaxing solution. For unclear reasons, the skinning procedure described above worked well for sheep EDL but not as well for rabbit psoas. The laser diffraction patterns from activated sheep fibres lasted longer without dispersion than the patterns from rabbit fibres. After experimenting with several skinning procedures, we adopted a method suggested by Dr Kevin Burton (personal communication) with some modifications. Strips of psoas measuring about 0.5 mm x 2 mm x 15 mm were tied to a glass rod and put in relaxing solution at 0 ~ C for 2 h followed by I h in a solution containing 25% glycerol and 75% relaxing solution. The tissue was then ready either for dissection into single fibres or storage for future dissection. If the flesh fibres were to be used immediately in experiments, a brief skinning procedure was required: the fibres were soaked in a solution containing 0.5% Triton X-IO0 and 99.5% relaxing solution for 30 min at 0 ~ C. If the muscle bundles were stored overnight or longer in the storage solution, the fibres could be used for experiments without additional skinning. The integrity of the sarcomere patterns lasted much longer with this new skinning procedure.
Dissection of single fibres A small bundle of fibres was dissected free from a muscle strip, The bundle was then separated into single fibre segments (approximately 2.5 mm long) in a dissecting tray containing 25% glycerol and 75% relaxing solution under a binocular microscope. The dissecting tray was maintained near 0 ~ C by circulating coolant. Aluminium foil clips (Ford
SEOW and F O R D
et al., 1977) were attached to the fibre segment at the ends, leaving approximately 1.5 mm of fibre length between the clips. The clipped fibres were either used immediately in experiments or stored in storage solution at - 2 0 ~ C for several days. APPARATUS The fibre was held horizontally in a covered trough into which precooled solution could be injected (Chiu et al., 1985). Both ends of the clipped fibre were connected to the wire hooks extending from the force-transducer and servo motor. The trough was kept at 1-2 ~ C by circulating coolant during the experiment. Solution change was accomplished by injecting solution into one end of the covered trough and removing solution from the other end by suction. The mechanical apparatus and associated control circuitry has been described in detail previously (Ford et al., 1977, 1990; Chiu et al., 1982, 1985; Seow & Ford, 1991b). Although the physical nature of the sarcomere length sensor has been described before (Ford et al., 199I) its use here requires further comment. Sarcomere length recording Sarcomere length was sensed by determining the position of the first diffracted order of a helium-neon laser using a linear position sensing photo-element (LSC-5D, United Detector Technology, Hawthorne, California). A set of lenses shaped the laser beam into a long slit focused on the fibre. The slit was approximately twice the length of the fibre and the ends of the slit were blocked by the aluminium foil clips used to hold the fibre. This arrangement provided that all of the sarcomeres were illuminated by a central portion of the slit. The position sensor gave a signal proportional both to light intensity and to the position of the centroid of the beam. To minimize spurious changes in apparent sarcomere length caused by changes in the intensity of the first order beam, the output of the sensor was divided by a signal proportional to intensity of the light reaching the sensor. This system would work perfectly if all of the light reaching the sensor derived from the first order beam. Unfortunately, some of the light is scatter from other orders, mainly the zero order. For this reason, the system cannot be depended on to give absolutely reliable measurements of sarcomere length. Additional precautions had to be taken to ensure the reliability of the system. Before the experiment, the system was calibrated using the 10th and 11th diffracted orders from an eyepiece reticle having 40 divisions per mm. At the outset of the experiments, a fibre was put in place and stretched so that no diffraction beam fell on the sensing element and the signal proportional to light intensity was set to zero. The magnitude of the intensity signal that developed when a fibre was positioned so that the first order beam fell on the sensor was then taken as a measure of the intensity of that beam. A running check in each experiment was then made by comparing the change in apparent sarcomere length with the change in overall muscle length during the late stages of steady state isotonic shortening. The muscle length record was converted to dimensions of/l m s -~ per half-sarcomere by dividing the overall length change by the number of half-sarcomeres. The overall muscle length and sarcomere length signals were measured at 50 and I42 ms after the onset
297
Damped passive recoil of skinned muscle fibre of an isotonic step. The change in the overall length signal over this interval was divided by the change in the sarcomere length to obtain a dimensionless ratio. This ratio was used as a scale factor to superimpose the sarcomere length records on the length records over the later periods of isotonic shortening (Fig. 1). In the present experiments, sets of isotonic steps, in which these length comparisons could be made, were done both before and after each set of slack test experiments, where no such comparisons could be made. If the average dimensionless ratio obtained with the two lowest loads in each set fell outside the limits of 0.1-1.1, the records were not used. In addition to being used to measure sarcomere length, the signal from this system was used to servo control sarcomere length during the isometric period before the length step in slack test experiments.
was activated. When a fibre was activated from a slightly shorter rest length (
Contribution of damped passive recoil to the measured shortening velocity of skinned rabbit and sheep muscle fibres C. Y. S E O W *
and L. E. F O R D
Section of Cardiology, Department of Medicine, University of Chicago, Chicago, IL 60637, USA Received 15 February 1991; revised and accepted 20 October 1991
Summary Maximum shortening velocities of skinned fibres from rabbit psoas and sheep extensor digitorum longus muscles were measured by the slack test and by extrapolating force-velocity curves to zero load. Both overall muscle velocity and sarcomere velocity were measured with each method. Maximum sarcomere velocity measured by the slack test was not significantly different from that assessed from the force-velocity curves (p > 0.1). Maximum overall muscle velocity measured from the slack test was significantly (p > 0.001) and substantially (62% rabbit, 83% sheep) greater than maximum sarcomere velocity. The difference is attributed to damped recoil of the series elastic elements contributing to the overall muscle velocity. The extent and time course of this damped recoil in isotonic steps was assessed from comparisons of overall muscle length and sarcomere length records during isotonic steps. When the records were shifted and scaled so that they superposed during the late stages of isotonic shortening, there was a substantial difference between the early parts of the records. This difference was reduced by about half in association with the step and the remaining half declined at a diminishing rate following the step, lasting longer with lower loads. This result is explained by about half of the series elastic element behaving as a viscoelastic element and half being undamped. With steps to the lowest isotonic loads, which averaged 6.7% of isometric force in sheep and 9.5% in rabbit, the total series elastic element recoil (both damped and undamped) averaged 3.4% and 2.7% of fibre segment length, respectively, in sheep and rabbit. The rapid series elastic element recoil at zero load, assessed from the slack test, was approximately 50% higher, indicating a substantial series compliance at low forces. The contribution of an additional, longer lasting, damped series elastic element recoil to the overall muscle velocity can explain the greater maximum velocity that is frequently found with the slack test.
Introduction Maximum shortening velocity of muscle is usually estimated by extrapolating the force-velocity curve to zero load, or by making the muscle go slack from isometric tension and measuring the time taken for force to begin to redevelop (Hill, 1970; Edman, 1979). The latter method, called slack test, has consistently yielded maximum velocities higher than those obtained from force-velocity curves in comparative studies (Edman, 1979; Julian & Moss, 1981; Moss, 1982; L~innergren et al., 1982; Goldman, 1983). As it is not likely that muscle *To whom correspondenceshouldbe addressed at: HospitalBox 249, University of Chicago Hospitals, 5841 South Maryland Avenue, Chicago, IL 60637, USA. 0142-4319 9
1992 Chapman & Hall
possesses distinct maximum velocities under different testing conditions, the discrepancy raises the question of the validity of the methods. In direct comparisons of the two methods, the disparity has ranged from 5% in studies of intact fibres (Edman, 1979) to 50% in skinned fibres (Julian & Moss, 1981). Our previous studies of the force velocity properties of skinned fibres showed that overall muscle velocity was substantially higher than sarcomere velocity at light loads during the early phase of isotonic shortening (Ford et al., 1991, Seow & Ford, 1991b). This difference was explained by damped recoil of the series elastic elements. In the present study we have assessed the effects of this damped recoil on measurements of maximum velocity determined by the two methods mentioned above. Some of these results
296 have been briefly presented in an abstract (Seow & Ford, 1991a).
Materials and methods MUSCLE FIBRE PREPARATION Animals A 55 kg sheep and several rabbits weighing about 2kg were used. Rabbits were first anaesthetized with intramuscular injections of 50 mg ketamine and 20 mg of xylazine, followed by an intracardiac injection of saturated KC1 solution to stop the heart. Muscle tissue from a sheep was obtained from a local abattoir immediately after the animal was slaughtered. Dissection and preparation of muscle bundles from the sheep Strips of extensor digitorum longus (EDL) were removed from the hind limbs of the sheep, tied to glass rods and kept overnight at 0~ in skinning solution (see SOLUTIONS).After the overnight skinning the tissue was soaked for I h in a solution containing 25% (by volume) glycerol and 75% relaxing solution (see SOLUTIONS) at 0 ~ C. Finally, the tissue was transferred to the storage solution containing equal portions of glycerol and relaxing solution. The pH of the storage solution was set to 6.5 at room temperature to provide a pH of 7.0-7.5 at the storage temperature of - 2 0 ~ C.
Dissection and preparation of muscle bundles from the rabbits Strips of psoas muscle were removed from the rabbits, tied to glass rods and kept at 0~ in relaxing solution. For unclear reasons, the skinning procedure described above worked well for sheep EDL but not as well for rabbit psoas. The laser diffraction patterns from activated sheep fibres lasted longer without dispersion than the patterns from rabbit fibres. After experimenting with several skinning procedures, we adopted a method suggested by Dr Kevin Burton (personal communication) with some modifications. Strips of psoas measuring about 0.5 mm x 2 mm x 15 mm were tied to a glass rod and put in relaxing solution at 0 ~ C for 2 h followed by I h in a solution containing 25% glycerol and 75% relaxing solution. The tissue was then ready either for dissection into single fibres or storage for future dissection. If the flesh fibres were to be used immediately in experiments, a brief skinning procedure was required: the fibres were soaked in a solution containing 0.5% Triton X-IO0 and 99.5% relaxing solution for 30 min at 0 ~ C. If the muscle bundles were stored overnight or longer in the storage solution, the fibres could be used for experiments without additional skinning. The integrity of the sarcomere patterns lasted much longer with this new skinning procedure.
Dissection of single fibres A small bundle of fibres was dissected free from a muscle strip, The bundle was then separated into single fibre segments (approximately 2.5 mm long) in a dissecting tray containing 25% glycerol and 75% relaxing solution under a binocular microscope. The dissecting tray was maintained near 0 ~ C by circulating coolant. Aluminium foil clips (Ford
SEOW and F O R D
et al., 1977) were attached to the fibre segment at the ends, leaving approximately 1.5 mm of fibre length between the clips. The clipped fibres were either used immediately in experiments or stored in storage solution at - 2 0 ~ C for several days. APPARATUS The fibre was held horizontally in a covered trough into which precooled solution could be injected (Chiu et al., 1985). Both ends of the clipped fibre were connected to the wire hooks extending from the force-transducer and servo motor. The trough was kept at 1-2 ~ C by circulating coolant during the experiment. Solution change was accomplished by injecting solution into one end of the covered trough and removing solution from the other end by suction. The mechanical apparatus and associated control circuitry has been described in detail previously (Ford et al., 1977, 1990; Chiu et al., 1982, 1985; Seow & Ford, 1991b). Although the physical nature of the sarcomere length sensor has been described before (Ford et al., 199I) its use here requires further comment. Sarcomere length recording Sarcomere length was sensed by determining the position of the first diffracted order of a helium-neon laser using a linear position sensing photo-element (LSC-5D, United Detector Technology, Hawthorne, California). A set of lenses shaped the laser beam into a long slit focused on the fibre. The slit was approximately twice the length of the fibre and the ends of the slit were blocked by the aluminium foil clips used to hold the fibre. This arrangement provided that all of the sarcomeres were illuminated by a central portion of the slit. The position sensor gave a signal proportional both to light intensity and to the position of the centroid of the beam. To minimize spurious changes in apparent sarcomere length caused by changes in the intensity of the first order beam, the output of the sensor was divided by a signal proportional to intensity of the light reaching the sensor. This system would work perfectly if all of the light reaching the sensor derived from the first order beam. Unfortunately, some of the light is scatter from other orders, mainly the zero order. For this reason, the system cannot be depended on to give absolutely reliable measurements of sarcomere length. Additional precautions had to be taken to ensure the reliability of the system. Before the experiment, the system was calibrated using the 10th and 11th diffracted orders from an eyepiece reticle having 40 divisions per mm. At the outset of the experiments, a fibre was put in place and stretched so that no diffraction beam fell on the sensing element and the signal proportional to light intensity was set to zero. The magnitude of the intensity signal that developed when a fibre was positioned so that the first order beam fell on the sensor was then taken as a measure of the intensity of that beam. A running check in each experiment was then made by comparing the change in apparent sarcomere length with the change in overall muscle length during the late stages of steady state isotonic shortening. The muscle length record was converted to dimensions of/l m s -~ per half-sarcomere by dividing the overall length change by the number of half-sarcomeres. The overall muscle length and sarcomere length signals were measured at 50 and I42 ms after the onset
297
Damped passive recoil of skinned muscle fibre of an isotonic step. The change in the overall length signal over this interval was divided by the change in the sarcomere length to obtain a dimensionless ratio. This ratio was used as a scale factor to superimpose the sarcomere length records on the length records over the later periods of isotonic shortening (Fig. 1). In the present experiments, sets of isotonic steps, in which these length comparisons could be made, were done both before and after each set of slack test experiments, where no such comparisons could be made. If the average dimensionless ratio obtained with the two lowest loads in each set fell outside the limits of 0.1-1.1, the records were not used. In addition to being used to measure sarcomere length, the signal from this system was used to servo control sarcomere length during the isometric period before the length step in slack test experiments.
was activated. When a fibre was activated from a slightly shorter rest length (
