T. Blangé

Tension development and calcium sensitivity in skinned muscle fibres of the frog

Calcium activated isometric tension development was measured in single skinned muscle fibres of the ileofibularis muscle of the frog. The experiments were carried out at 5°C, pH=6.9, 1 mM free Mg2+ and an ionic strength of 160 mM. A Hill curve was fitted to the isometrically developed tension at different Ca2+ concentrations by means of a non-linear least mean square approximation. At a sarcomere length of 2.15 μm, the Ca2+ concentration for half maximum tension (K) was 1.6 μM. This Ca2+ concentration decreased with increasing sarcomere length; at 2.7 μm, K was 1.1 μM and at 3.1 μm, K was 0.9 μM. Therefore, Ca sensitivity is increased at larger sarcomere lengths. Consequently, the optimal sarcomere length for tension development shifted to larger values when the Ca2+ concentration was lowered. Osmotic compression of the fibre at 2.15 μm by means of 5% Dextran also caused an increase in Ca sensitivity (K was 1.0 μM). At 2.7 μm, addition of 5% Dextran hardly affected the Ca sensitivity. The possible role of the interfilament spacing in the explanation of these results discussed.

Elasticity as an Expression of Cross-Bridge Activity in Rat Muscle

Summary1.Rat soleus muscles (SOL) were isolated and the force-shortening curves were determined during isometric maximal tetani. These were measured by applying sudden shortenings of different magnitudes to one end of the muscle while recording the resulting drop in tension.2.From the force-shortening curve a stiffness-force curve was derived. Then a stiffness-force curve was measured by applying small shortenings (20 μm) to the muscle when at different lower stimulation frequencies it was exerting less than maximal tetanic force.3.It appeared that the two curves, taken from the same preparations, differed consistently. It was therefore concluded that muscle stiffness at a certain force depends on the way by which this force was reached. This conclusion is shown to fit very well to a sliding filaments model like the one Huxley proposed in 1957.4.The sudden shortenings applied to the muscle gave rise to a travelling wave in the muscle. The resulting tension transients could be explained by assuming that muscle behaves as an homogeneous body with distributed mass, stiffness and damping.

Tension responses of frog sartorius muscle to quick ramp-shaped shortenings and some effects of metabolic inhibition.

AbstractThe dynamic properties of the contractile mechanism of skeletal muscle were investigated in electrically stimulated sartorius muscle of the frog at 0°C by analysis of the force responses to quick changes in length. 1.The tension responses to ramp-shaped shortenings at different rates of shortening have been recorded. The tension course during the ramp is described in terms of a damped elastic element. In addition a maximum value for an additional series elastic element is estimated.2.The tension responses to pulse-shaped shortenings have been recorded. At least 15ms after the pulse a minimum in tension is found, which depends on amplitude and duration of the pulse. This effect is optimal at a pulse duration of about 6ms. It is concluded that within 15ms after the pulse detachment of crossbridges occurs.3.Muscles were incubated with the metabolic inhibitors IAA and FAA or IAA and FDNB. After repeated stimulation, but before the muscle passes into the rigor state, the fast recovery and the plateau in the responses to quick shortenings are either diminished or abolished, depending on the amplitude of the shortening. This can be explained by assuming that in the unpoisened muscle, ATP enables the crossbridges to exert temporarily a larger force.

Elastic properties of relaxed, activated, and rigor muscle fibers measured with microsecond resolution

Tension responses due to small and rapid length changes (completed within 40 microseconds) were obtained from skinned single-fiber segments (4- to 7-mm length) of the iliofibularis muscle of the frog incubated in relaxing, rigor, and activating solution. The fibers were skinned by freeze-drying. The first 500 microseconds of the responses for all three conditions could be described with a linear model, in which the fiber is regarded as a rod composed of infinitesimally small identical segments, containing an undamped elastic element, two damped elastic elements and a mass in series. An additional damped elastic element was needed to describe tension responses of activated fibers up to the first 5 ms. Consequently phase 1 and phase 2 of activated fibers can be described with four apparent elastic constants and three time constants. The results indicate that fully activated fibers and fibers in rigor have similar elastic properties within the first 500 microseconds of tension responses. This points either to an equal number of attached cross-bridges in rigor and activated fibers or to a different number of attached cross-bridges in rigor and activated fibers and nonlinear characteristics in rigor cross-bridges. Mass-shift measurements obtained from equatorial x-ray diffraction patterns support the latter possibility.

Weakly attached cross-bridges in relaxed frog muscle fibers

Tension responses due to small, rapid length changes (completed within 40 microseconds) were obtained from skinned single frog muscle fiber segments (4-10 mm length) incubated in relaxing and rigor solutions at various ionic strengths. The first 2 ms of these responses can be described with a linear model in which the fiber is regarded as a rod, composed of infinitesimally small, identical segments, containing one undamped elastic element and two or three damped elastic elements and a mass in series. Rigor stiffness changed less than 10% in a limited range, 40-160 mM, of ionic strength conditions. Equatorial x-ray diffraction patterns show a similar finding for the filament spacing and intensity ratio I(11)/I(10). Relaxed fibers became stiffer under low ionic strength conditions. This stiffness increment can be correlated with a decreasing filament spacing and (an increased number of) weakly attached cross-bridges. Under low ionic strength conditions an additional recovery (1 ms time constant) became noticeable which might reflect characteristics of weakly attached cross-bridges.

The length dependency of calcium activated contractions in the femoral artery smooth muscle studied with different methods of skinning

The relationship between the calcium concentration and the isometric tension obtained with different techniques of skinning provides information on the biochemical events of contraction in vascular smooth muscle. Muscle preparations of the rabbit femoral artery were skinned with triton X-100, saponin, β-escin and α-toxin and the relationship between the calcium concentration and isometric tension was determined at different preparation lengths. We determined the calcium sensitivity as a function of muscle length with different techniques of skinning. At a pCa of 6.0, triton X-100 skinned smooth muscle of the femoral artery generated 50% of the maximal tension. In α-toxin skinned preparations, this calcium sensitivity was shifted to a pCa of 5.6. The sensitivity of the saponin and β-escin skinned preparations were in between those of the triton X-100 and the α-toxin skinned preparations. The cooperativity of the regulation of contraction varied among the differently skinned preparations between 3 (α-toxin) and 6 (triton X-100). The relationships between the calcium concentration and the isometric tension of the differently skinned preparations up to the optimal length for tension generation did not exhibit any length dependency. The length tension relationship, obtained from the maximal response at the highest calcium concentration is in line with that from other studies. The presence of intracellular proteins and membranes affects the regulation of contraction in smooth muscle of the femoral artery.

The effect of actin filament compliance on the interpretation of the elastic properties of skeletal muscle fibres.

Recently, X-ray diffraction studies provided direct evidence for an appreciable length change in the actin filament upon activation. This finding has profound implications on the interpretation of the elastic properties of skeletal muscle fibre. In this study we determined the compliance of the actin filament during activation, using the data obtained previously from quick stretch and release experiments on skeletal muscle fibres of the frog. The effects of filament compliance are demonstrated clearly in the elastic properties of partially activated fibres. The low- frequency elasticity increases linearly with tension, reflecting an increase in the number of force-producing cross-bridges. At higher frequencies, this linearity is lost. In this study we describe the data consistently in terms of a cross-bridge stiffness increasing linearly with tension and a constant Youngs modulus for the actin filament of 44 MN m−2. This corresponds to a compliance of 23 pm μm−1 per kN m−2 tension developed. Using this value for the actin filament Youngs modulus, its contribution to the elastic properties of skeletal muscle fibre of the frog is considered in rigor and relaxation. The filament compliance hardly affects the overall elasticity of the musle fibre in relaxation. In contrast, it contributes to a large extent to the overall elasticity in rigor. Taking account of the filament compliance, we find that the Youngs modulus in rigor exhibits an increase from 14 MN m−2 at frequencies below 500 Hz to 55 MN m−2 above 40 kHz

Tension responses to rapid length changes in skinned muscle fibres of the frog

Tension responses due to rapid length changes completed in 50 and 150 μs were obtained from activated skinned single fibres of the ileofibularis muscle of the frog. The natural frequency of the force transducer was about 50 kHz. The length changes ranged between −1% and +0.5% of the fibre segment length. The sarcomere length was adjusted to 2.15 μm. The temperature was maintained at 2.5° C. The transmission velocity estimated from these recordings obtained on fibre segments with different length was 230 m/s in fully activated segments and 112 m/s in relaxed segments. The initial part of the responses during the length changes consisted of an abrupt change in tension reaching an extreme value T1, which depended on the amplitude as well as the duration of the length change. A partial rapid recovery towards a plateau occurred after the length change. The reciprocal half-time of this recovery increased with the amplitude of the displacement both for stretches as well as releases up to about 5 nm/half sarcomere.

Cross-bridge stiffness in Ca2+-activated skinned single muscle fibres

Tension transients, in response to small and rapid length changes (completed within 40 μs), were obtained from skinned single frog muscle fibres incubated in activating solutions with varying concentrations of Ca2+. The first 2 ms of these transients were described by a linear model in which the fibre is regarded as a rod composed of infinitesimally small, identical segments containing a mass, one undamped elastic element and in the case of relaxed fibres two damped elastic elements in series, or in the case of activated fibres three such elastic elements in series. The stiffness of activated fibres, expressed in elastic constants or apparent elastic constants, increased with increasing concentrations of Ca2+. All the damped elastic constants that were necessary to describe the tension responses of activated fibres were proportional to isometric tension. However, the undamped elastic constant did not increase linearly with increasing isometric tension. Equatorial X-ray diffraction patterns were obtained from single frog muscle fibres under similar conditions as under which the tension transients were obtained. The filament spacing (d10)of Ca2+-activated single frog muscle fibres decreased with increasing isometric force, whereas the intensity ratio (I11/I10)increased linearly with increasing isometric force. From experiments in which dextran (MW 200000 Da) was added, it followed that such a change in filament spacing would modify passive stiffness. The d10value of relaxed fibres decreased and stiffness increased with increasing concentrations of the polymer dextran, whereas I11/I10remained constant. The relation of stiffness and filament spacing with concentration of dextran was used to eliminate the effect of decreased filament spacing on stiffness of activated fibres. After correction for changes in filament spacing the undamped complicance C1, normalized to tension, was not constant, but increased with increasing isometric tension. If we assume that isometric tension is proportional to the number of force generating cross-bridges, this means that only part of the undamped complicance of activated fibres is located in the crossbridges.

The effect of length on the sensitivity to phenylephrine and calcium in intact and skinned vascular smooth muscle.

The length dependency of the sensitivity to activators of the smooth muscle of different blood vessels is not yet fully understood. Muscle preparations of the aorta, the femoral artery and the portal vein of the rabbit were investigated for the length dependency of the sensitivity to phenylephrine and calcium in both intact and triton X- 100 skinned preparations. For intact smooth muscles we found that at increased preparation length, the sensitivity of contraction was increased. The femoral artery showed the largest effect and the portal vein the smallest. In the skinned preparations of the three preparations the calcium sensitivity was not dependent on the preparation length. We conclude that the changes of the sensitivity in intact preparations are not caused by changes of the calcium sensitivity of the contractile proteins.