Stuart R. Taylor

Stiffness and force in activated frog skeletal muscle fibers

Single fibers, isolated intact from frog skeletal muscles, were held firmly very near to each end by stiff metal clasps fastened to the tendons. The fibers were then placed horizontally between two steel hooks inserted in eyelets of the tendon clasps. One hook was attached to a capacitance gauge force transducer (resonance frequency up to approximately 50 kHz) and the other was attached to a moving-coil length changer. This allowed us to impose small, rapid releases (complete in less than 0.15 ms) and high frequency oscillations (up to 13 kHz) to one end of a resting or contracting fiber and measure the consequences at the other end with fast time resolution at 4 to 6 degrees C. The stiffness of short fibers (1.8-2.6 mm) was determined directly from the ratio of force to length variations produced by the length changer. The resonance frequency of short fibers was so high (approximately 40 kHz) that intrinsic oscillations were not detectably excited. The stiffness of long fibers, on the other hand, was calculated from measurement of the mechanical resonance frequency of a fiber. Using both short and long fibers, we measured the sinusoids of force at one end of a contracting fiber that were produced by relatively small sinusoidal length changes at the other end. The amplitudes of the sinusoidal length changes were small compared with the size of step changes that produce nonlinear force-extension relations. The sinusoids of force from long fibers changed amplitude and shifted phase with changes in oscillation frequency in a manner expected of a transmission line composed of mass, compliance, and viscosity, similar to that modelled by (Ford, L. E., A. F. Huxley, and R. M. Simmons, 1981, J. Physiol. (Lond.), 311:219-249). A rapid release during the plateau of tetanic tension in short fibers caused a fall in force and stiffness, a relative change in stiffness that putatively was much smaller than that of force. Our results are, for the most part, consistent with the cross-bridge model of force generation proposed by Huxley, A. F., and R. M. Simmons (1971, Nature (Lond.), 213:533-538). However, stiffness in short fibers developed markedly faster than force during the tetanus rise. Thus our findings show the presence of one or more noteworthy cross-bridge states at the onset and during the rise of active tension towards a plateau in that attachment apparently is followed by a relatively long delay before force generation occurs. A set of equations is given in the Appendix that describes the frequency dependence of the applied sinusoid and its response. This model predicts that frequency dependent changes can be used as a measure of a change in stiffness.

Skeletal muscle sarcoplasmic reticulum in porcine malignant hyperthermia

To examine the function of sarcoplasmic reticulum (SR) in malignant hyperthermia, SR was isolated from semitendinosus muscle of normal and genetically susceptible Poland China swine. Determinations included rate of calcium binding (oxalate absent), rate and capacity of calcium uptake (oxalate present), and spontaneous calcium release (in the absence of ionic depolarization or calcium) with and without halothane, using the millipore filtration technique. Rate of calcium binding, and rate and capacity of calcium uptake were decreased, and spontaneous calcium release was greater in SR fragments from susceptible swine as compared to those from normal swine. Halothane 0.5% slightly increased the rate of calcium binding in susceptible and normal SR. Above 1%, halothane decreased calcium binding rate, and uptake rate and capacity, and increased calcium release similarly in susceptible and normal SR. These differences in SR function were insufficient to explain the etiology of malignant hyperthemia, nor did the effect of halothane account for its triggering action.

High-speed video imaging and digital analysis of microscopic features in contracting striated muscle cells

The rapid motion of microscopic features such as the cross striations of single contracting muscle cells are difficult to capture with conventional optical microscopes, video systems, and image processing approaches. An integrated digital video imaging microscope system specifically designed to capture images from single contracting muscle cells at speeds of up to 240 Hz and to analyze images to extract features critical for the understanding of muscle contraction is described. This system consists of a brightfield microscope with immersion optics coupled to a high-speed charge-coupled device (CCD) video camera, super-VHS (S-VHS) and optical media disk video recording (OMDR) systems, and a semiautomated digital image analysis system. Components are modified to optimize spatial and temporal resolution to permit the evaluation of submicrometer features in real physiological time. This approach permits the critical evaluation of the magnitude, time course, and uniformity of contractile function throughout the volume of a single living cell with higher temporal and spatial resolutions than previously possible.

A transient and a persistent calcium release are induced by chlorocresol in cultivated mouse myotubes.

Abstract The effect of 4-chloro-m-cresol (4-CmC), a stabilizing agent used in commercial preparations of the muscle relaxant succinylcholine, on intracellular free calcium levels in cultivated mouse myotubes was studied. Calcium signals were monitored with an inverted microscope equipped for fluorescence photometry using fura-2 as the calcium indicator. Upon bath application of 500 µM 4-CmC for 90 s, two separate calcium signals, a transient and a sustained one, could be regularly discriminated. First, with a delay of 2 s, the intracellular calcium concentration increased from 41±13 to 541±319 nM, peaked after 2–5 s and declined within 10 s to nearly resting values (n=36). Then, after a delay of up to 20 s, intracellular calcium rose quickly again to almost the same value and stayed elevated as long as the drug was applied. Upon drug removal, intracellular calcium rapidly decreased to a new level that was always slightly higher than the original base line. At 250 µM 4-CmC, the response was small, whereas at 500 µM it was at its maximum. Thus, the concentration-response curve was very steep. Replacement of extracellular calcium by EGTA and application of calcium channel blockers revealed that, for both the transient and the sustained response, calcium was released from intracellular stores. Pre-treatment with thapsigargin (0.1 µM) or ryanodine (10 µM) abolished both signal components. Repeated short-term applications of 4-CmC suggest that the two components may arise from different systems.

The Kinetics of Cross-Bridge Attachment and Detachment Studied by High Frequency Stiffness Measurements

Muscle fiber stiffness, supposedly an indication of attached cross-bridges, was measured throughout tetanic contraction and subsequent relaxation. Stiffness increased at a rate faster than the development of force during the rise of tetanic contraction and decreased more slowly than force during relaxation. One explanation for these results is that long-lived cross-bridge states may exist between attachment, force generation and detachment.

Volume Changes During Contraction of Isolated Frog Muscle Fibers

A microscope objective and electronic imaging system were used to determine how isolated frog skeletal muscle fibers adjust their volume during an isometric tetanus. Cross-sectional area and volume of the middle third of a fiber increased rapidly with the development of active tension, which indicates that contraction produced components of force perpendicular to the long axis. The extreme ends are known to shorten whether or not the middle of a fiber is isometric or stretched. Shortening of the ends may shift water towards the middle, which could account for the volume changes we observed. The cytoskeletal matrices of muscle evidently adjust rapidly during contraction to maintain a dynamic equilibrium between the axial and radial forces that stabilize the whole cell. The Z disks have been shown to expand during active, but not passive, tension development. Z disks might be the elastic elements of the muscle cytoskeleton primarily involved in rapid balancing of the radial components of active force.

Changes in Intracellular Ca2+ Induced by Shortening Imposed during Tetanic Contractions

Calcium transients, monitored by aequorin, and force were recorded simultaneously during tetanic contractions of isolated frog skeletal muscle fibers. Quick length changes were applied to the fibers during contractions at sarcomere lengths on the descending limb of the length-tension relationship. Previous experiments showed that regulatory Ca2+ binding sites are apparently saturated during a plateau of tetanic force development at these sarcomere lengths. However, quick releases of greater than 4 to 5% of fiber length produced a momentary fall in the calcium transient that followed a time course similar to the redevelopment of force. The fall in the Ca2+ transient after a release was maximum at striation spacings about half way along the descending limb (2.6-2.7 microns), which suggests it is not related to an increase in the number of Ca2+ binding sites distributed uniformly along the filaments. The effect was absent or barely detectable when highly stretched fibers were released during contraction. The fall in the Ca2+ transient was unrelated to the time during a tetanus that a release was made or to the velocity of the release. One explanation of these results is that complexes between actin and myosin are broken by a sudden reduction of length, and as they reform during the recovery of force the affinity of troponin for Ca2+ increases. Quick stretch had no effect on the rapid decay of Ca2+ transients, but stretch increased peak force and slowed relaxation for almost a second after the end of stimulation. Evidently the decrease in the rate of relaxation produced by stretch is unrelated to changes in the amount of Ca2+ released or the rate of Ca2+ removal, which supports suggestions that the kinetics of muscle relaxation are determined by more than one mechanism. The apparent increase in the overall duration of mechanical activity after stretch probably results from the longitudinal inhomogeneity in the duration of activity - known to occur during relaxation - coupled with the decreased compliance of stretched fibers.

High Speed Striation Pattern Recognition In Contracting Cardiac Myocytes

The understanding of muscle contraction and relaxation requires the quantitation of movement at the sub-micron level in living cells. Two complementary non-RS-170 imaging systems used for authentic real time measurement of contractile dynamics are described and compared. Images from isolated skeletal or cardiac muscle cells are projected by an optical microscope onto single line or area charge-coupled device (CCD) photodiode arrays. These data are digitized and stored for subsequent image processing and analysis. The inherently low contrast muscle striation patterns are enhanced and their rapid movement measured with an accuracy at least an order of magnitude greater than traditional limits of optical resolution. The features of each image format are complementary and when combined provide the maximum overall information in time and space.

Three-dimensional reconstruction of active muscle cell segment volume from two-dimensional optical sections

An ultramicroscope coupled to a square-aspect-ratio sensor was used to image the dynamic geometry of live muscle cells. Skeletal muscle cells, dissected from frogs, were suspended in the optical axis and illuminated from one side by a focused slit of white light. The sensor detected light scattered at 90 degrees to the incident beam. Serial cross-sections were acquired as a motorized stage moved the cell through the slit of light. The axial force at right angles to the cross- sections was recorded simultaneously. Cross-sections were aligned by a least-squares fit of their centroids to a straight line, to correct for misalignments between the axes of the microscope, the stage, and the sensor. Three- dimensional volumes were reconstructed from each series and viewed from all directions to locate regions that remained at matching axial positions. The angle of the principal axis and the cross-sectional area were calculated and associated with force recorded concurrently. The cells adjusted their profile and volume to remain stable against turning as contractile force rose and fell, as predicted by the law of conservation of angular momentum.

Reduced defocus degradation in a system for high-speed three-dimensional digital microscopy

Most low-light-level 3D bio-imaging systems use sensors with high spatial resolution but relatively low temporal resolution. The authors aim to measure events in skeletal muscle cells that include (1) changes in cross-sectional area during contraction and (2) regions of contracting cells illuminated by Ca2+-activated photoproteins compared with those that are dark or partly lit. This paper outlines some limits of the systems ability to detect these events.