Cross-bridge kinetics of rabbit single extraocular and limb muscle fibers.

PURPOSE: To gain insights into the functional significance of myosin heavy-chain (MyHC) heterogeneity by comparing the mechanical kinetic properties of single rabbit extraocular muscle (EOM) fibers with those of limb fibers. EOMs are known to contain developmental and EOM-specific MyHCs in addition to those present in limb muscles, and MyHCs profoundly influence muscle mechanics. METHODS: Isometric cross-bridge kinetics were analyzed in Ca(2+)-activated single glycerinated fibers from rabbit EOM and limb fast and slow muscles at 15 degrees C by means of mechanical perturbation analysis. The plots of stiffness and phase against frequency display a characteristic frequency, f(min), at which stiffness is minimum, and phase shift is zero. The value of f(min) is independent of Ca(2+) or force level but reflects the kinetics of cross-bridge cycling. RESULTS: Analysis of 121 limb fast fibers gave f(min) values ranging from 10 to 26 Hz. f(min) for the 10 slow soleus fibers was 0.5 Hz. Analysis of 170 EOM fibers gave f(min) values in the range for fast limb fibers, but in addition yielded f(min) values below (4-9 Hz) and above (27-33 Hz) this range. CONCLUSIONS: The wider range of mechanical kinetic characteristics in EOM fibers compared with limb fibers is likely due to the expression of developmental (low f(min)) and EOM-specific (high f(min)) MyHCs in addition to isoforms present in adult limb muscles. The considerable diversity of functional characteristics in EOM fibers is likely to be important for rotating the eyeball at various speeds during tracking and for executing saccades over a wide range of angles.

Relationships between isometric and isotonic mechanical parameters and cross-bridge kinetics.

1. Isometric and isotonic measures have been widely used both to characterize muscular contraction in striated muscle and to compare the mechanical properties of striated muscle types for a wide variety of species. The interrelationship of these measures and their interpretation, in terms of underlying cross-bridge mechanisms, requires clarification. 2. We demonstrate that a strain-dependent three-state model can simulate these isometric and isotonic mechanics and offer a unified explanation for these mechanics in terms of cross-bridge kinetics. 3. Our exploration reveals the partial view each individual measure of mechanics provides and emphasizes the complementary insights the various isometric and isotonic measures give of the underlying cross-bridge kinetics.

Mechanism of action of endothelin in rat cardiac muscle: cross-bridge kinetics and myosin light chain phosphorylation.

1. The molecular mechanism of inotropic action of endothelin was investigated in rat ventricular muscle by studying its effects on characteristics of isometric twitch, barium-induced steady contracture and the level of incorporation of 32Pi into myosin light chain 2. 2. Exposure of rat papillary muscle to endothelin caused an increase in isometric twitch force but did not alter the twitch-time parameters. 3. Endothelin did not significantly change the maximum contracture tension but did cause an increase in contracture tension at submaximal levels of activation, without changes in the tension-to-stiffness ratio and kinetics of attached cross-bridges. Kinetics of attached cross-bridges were deduced during steady contracture from complex-stiffness values, and in particular from the frequency at which muscle stiffness assumes a minimum value, fmin. Endothelin did not alter fmin. 4. Endothelin caused an increase in the level of incorporation of 32Pi into myosin light chain 2 without a concurrent change in the level of incorporation of 32Pi into troponin I. 5. We conclude that the inotropic action of endothelin is not due to an increase in the kinetics of attached cross-bridges, nor due to a change in the force per unit cross-bridge, but may result from an increased divalent cation sensitivity caused by elevated myosin light chain 2 phosphorylation, resembling post-tetanic potentiation in fast skeletal muscle fibres.

Influence of myosin isoforms on tension cost and crossbridge kinetics in skinned rat cardiac muscle.

1. In attempting to consolidate the role of ventricular isomyosins in regulating the contractility of the myocardium, actomyosin ATPase and crossbridge kinetics were obtained at 24 degrees C in chemically skinned isometrically contracting cardiac muscles containing V1 and V3 isomyosins. 2. The ATPase activity was measured at various levels of Ca2+ activation by the enzymatic coupling of ATP hydrolysis with the conversion of NADH to NAD+. The crossbridge kinetics were inferred from small-amplitude perturbations of muscle length and muscle tension, and characterized by the frequency-domain parameter fmin. 3. The ATPase rates of V1 and V3 muscles obtained at various levels of Ca2+ activation were plotted against the corresponding proportional tensions. The ATPase vs tension plots were linear with slopes of 4.92 nmol/min-1 per mm per mN and 1.98 nmol/min-1 per mm per mN, respectively for, V1 and V3 muscles. Individual calculations of ATPase-to-tension ratios (nmol/min-1 per mm per mN) gave corresponding averages of 4.98 +/- 0.12 (s.e.m., n = 12) and 2.16 +/- 0.12 (s.e.m., n = 10). The myosin isoform induced proportional change in tension cost was accompanied by a similar change in fmin (4.1 +/- 0.1 Hz and 1.95 +/- 0.03 Hz, means +/- s.e.m., for V1 and V3 muscles, respectively). 4. The observations and other published kinetic data are discussed in the context of models of crossbridge cycling. It is suggested that the tension economy of V3 muscle arises principally from an increase in the fraction of time, during the crossbridge cycle, when the crossbridge is exerting force.

Effects of dibutyryl cyclic AMP, ouabain, and xanthine derivatives on crossbridge kinetics in rat cardiac muscle.

In a previous communication, we showed that beta-adrenergic stimulation of cardiac muscles was associated with an increase in the rate of cycling of crossbridges as measured by perturbation analysis in the frequency domain. In this analysis, the frequency at which dynamic stiffness is a minimum (fmin) is taken as a measure of the rate of crossbridge cycling. In this paper, we test the hypothesis that the beta-adrenergic receptor-induced increase in crossbridge cycling rate is mediated by elevation of the intracellular level of cyclic AMP. The approach taken is to compare the effects on fmin in rat papillary muscles during Ba(2+)-activated contractures of 1) an agonist of cyclic AMP that can easily penetrate the cell, namely, dibutyryl cyclic AMP, 2) agents that block cyclic AMP phosphodiesterase, namely, the xanthine derivatives isobutylmethylxanthine and caffeine, and 3) an inotropic agent that does not affect the intracellular level of cyclic AMP, namely, ouabain. Our results showed that dibutyryl cyclic AMP at a dose of 5 mM has the same actions as beta-adrenergic stimulation: it potentiated the isometric twitch force, reduced the time to peak tension and time to half relaxation, and shifted fmin by a factor of 1.8 +/- 0.1 (n = 5). Isobutylmethylxanthine at up to 1.1 mM also acted in the same manner, increasing fmin by a factor of 1.8 +/- 0.2 (n = 6), but ouabain, at a dose (0.03 mM) sufficient to potentiate twitch force by 40 +/- 2% (n = 4), was without effect on the time course of the twitch nor was fmin changed (n = 4). Our findings support the hypothesis that a beta-adrenergic receptor-mediated increase in crossbridge cycling rate is due to an increase in intracellular cyclic AMP level and illustrate the usefulness of the frequency domain analysis approach in the analysis of the mechanism of action of inotropic agents.

The role of calcium ions in the activation of rabbit psoas muscle.

This study tested the effects of free Ca++ on both the small-amplitude mechanical behaviour (dynamic stiffness and phase between 1 and 500 Hz) and the large-scale filament-sliding behaviour (Vmax) of single fibres of chemically-activated glycerol-extracted rabbit psoas muscle. Small-amplitude vibrations (0.1% peak-to-peak of initial length L0) were used to elicit fw, the frequency for maximum oscillatory work-production per cycle. The unloaded contraction velocity Vmax was measured during the same contractions, using the slack-test method with shortening length steps of up to 10% L0. These produced nonlinear length-time plots demonstrating that the unloaded contraction velocity was not constant as contraction progressed, but fell with time. This behaviour was approximated by two velocities, V1 the velocity observed for about the first 15 ms and V2 the velocity after this time. V1 and V2 were found to have different sensitivities to Ca++. The value of V2 fell as the level of [Ca++] was reduced, and was linearly proportional to the active tension over the range 0.2 Pmax to Pmax (where Pmax is the isometric tension for saturating amounts of Ca++). In contrast to this V1 remained insensitive to changes in [Ca++] for levels of activation corresponding to active tensions ranging from Pmax to 0.6 Pmax, and then fell as the level of activation was further reduced. It was found that the level of [Ca++] did not affect the magnitude of fw over the range of concentrations yielding active tensions from 0.2 Pmax to Pmax. These results are discussed in terms of the kinetic processes underlying transient readjustments to perturbations from isometric equilibrium.

Adrenaline increases the rate of cycling of crossbridges in rat cardiac muscle as measured by pseudo-random binary noise-modulated perturbation analysis.

The mechanism of action of adrenaline on cardiac contractility in rat papillary muscles containing V1 and V3 isomyosins was analyzed during barium-activated contractures at 25 degrees C by frequency domain analysis using pseudo-random binary noise-modulated perturbations. The analysis characterizes a frequency (fmin) at which dynamic stiffness of a muscle is a minimum, a parameter that reflects the rate of cycling of crossbridges. We have previously shown that fmin for V1- and V3-containing papillary muscles were 2.1 +/- 0.2 Hz (mean +/- SD) (n = 10) and 1.1 +/- 0.2 Hz (n = 8), respectively, and that these values were independent of the level of activation. The present study's goal was to determine whether the inotropic action of adrenaline was associated with an increased rate of crossbridge cycling. The results show that a saturating dose of adrenaline increased fmin in V1 hearts by 49 +/- 2% (n = 11). The action on V3 hearts was significantly less; the increase in fmin was 26 +/- 2% (n = 6). The increase in fmin for V1 hearts was shown to be sensitive to the beta-blocking agent propranolol. These results suggest that adrenaline significantly increases the rate of crossbridge cycling by a beta-receptor-mediated mechanism. We conclude that the increased contractility of the heart in the presence of adrenaline arises not only from more complete activation of the contractile proteins but also from the increased rate at which each crossbridge can transduce energy.

Tension responses of muscle to n-step pseudo-random length reversals: a frequency domain representation.

A high resolution, time efficient method is described for determining the complex modulus of activated striated muscle. In this method the applied oscillation of muscle length takes the form of pseudo-random binary noise, PRBN. As a time-domain signal, PRBN is an extension of the double step approach to include n steps; as a frequency-domain signal, PRBN has the properties of quasi-white noise. Fourier analysis of the PRBN length oscillations and the resulting interrupted tension transients gives rise to the complex modulus values. PRBN provides a practical demonstration of the conceptual link between time and frequency domain descriptions of strain sensitive dynamics. The method is demonstrated with intact rat papillary muscle, and glycerol extracted rabbit psoas muscle.

Influence of V1 and V3 isomyosins on the mechanical behaviour of rat papillary muscle as studied by pseudo-random binary noise modulated length perturbations.

Experiments were done on four-week-old rats, containing biochemically verified V1 only, and thyroidectomized adult rats, treated with propylthiouracil, verified to contain V3 only. Contracture tension was induced in isolated papillary muscles either by high potassium solution or 0.5 mmol l-1 Ba2+. Small amplitude length perturbations with peak-to-peak value not exceeding 0.15% L0 were applied to the activated muscle. Both the applied length perturbations and the corresponding resulting force changes were analysed by computer for dynamic stiffness and phase values. In order to reduce data acquisition time, pseudo-random binary noise length changes, rather than the conventional sinusoidal length changes, were used. The plot of the dynamic stiffness against frequency displays a minimum, akin to a resonance phenomenon. The frequency, fmin, at which this resonance occurs, reflects crossbridge kinetics. It was found that the fmin values for the two types of papillary muscles differed by a factor of two. Experiments were also done on chemically skinned muscles containing V1 or V3 isomyosin activated by different concentrations of either barium or calcium ions. It was found that fmin values of skinned fibres were higher than those obtained from intact fibres. However, for each type of muscle the fmin was independent of the activator used as well as the level of activation. The ratio of fmin for V3 to that for V1 remained the same as for intact preparations. We conclude that the difference in mechanical parameters did not arise a possible difference in excitation-contraction coupling mechanism, but rather is a difference in the dynamic properties of the two types of crossbridges.

Frequency-domain study of the mechanical response of living striated muscle.

Small-amplitude sinusoidal displacements, in the frequency range 4-100 Hz, were applied to intact whole frog sartorius muscle whilst in a state of tetanus. At low frequencies the muscle was observed to do oscillatory work, while at higher frequencies it tended towards elastic behaviour. Frequency-response plots obtained were compared with those from other muscle preparations. Results were interpreted in terms of mechano-chemical transduction properties of muscle.