Time Course Of Force Development Research Articles

Filament lattice changes in smooth muscle assessed using birefringence

The long functional range of some types of smooth muscle has been the subject of recent study. It has been proposed that the muscle filament lattice adapts to longer lengths by placing more filaments in series and that lattice plasticity is facilitated by myosin filament evanescence, with filaments dissociating during relaxation and reforming upon activation. Support for these dynamic changes in the filament lattice has been provided partly by changes in contractile parameters at different times in the contraction-relaxation cycle at different lengths. If the changes in contractile parameters result from filament formation and dissociation, these structural changes must occur on the time scale of tension development and relaxation. To assess whether thick-filament formation could account for the contractile changes, we measured birefringence continuously during activation and relaxation and compared these optical changes with the time course of force development and relaxation. Birefringence is a well-known property of muscle; striations in skeletal and cardiac muscle result from the A-bands being anisotropic, i.e., birefringent, and it is now known that this optical property is due to the presence of myosin thick filaments in the A-bands. Thus, the strength of birefringence is expected to represent the density of thick filaments. Here, we describe the principle of the method, the techniques for recording the optical signals, some initial results, and discuss the interpretation of results and some limitations of the method.

Influence of ionic strength on the time course of force development and phosphate release by dogfish muscle fibres

We measured the effects of ionic strength (IS), 200 (standard) and 400 mmol l(-1) (high), on force and ATP hydrolysis during isometric contractions of permeabilized white fibres from dogfish myotomal muscle at their physiological temperature, 12 degrees C. One goal was to test the validity of our kinetic scheme that accounts for energy release, work production and ATP hydrolysis. Fibres were activated by flash photolysis of the P(3)-1-(2 nitrophenyl) ethyl ester of ATP (NPE-caged ATP), and time-resolved phosphate (P(i)) release was detected with the fluorescent protein MDCC-PBP, N-(2[1-maleimidyl]ethyl)-7-diethylamino-coumarin-3-carboxamide phosphate binding protein. High IS slowed the transition from rest to contraction, but as the fibres approached the isometric force plateau they showed little IS sensitivity. By 0.5 s of contraction, the force and the rate of P(i) release at standard and high IS values were not significantly different. A five-step reaction mechanism was used to account for the observed time courses of force and P(i) release in all conditions explored here. Only the rate constants for reactions of ATP, ADP and P(i) with the contractile proteins varied with IS, thus suggesting that the actin-myosin interactions are largely non-ionic. Our reaction scheme also fits previous results for intact fibres.

The ATP hydrolysis and phosphate release steps control the time course of force development in rabbit skeletal muscle

The time course of isometric force development following photolytic release of ATP in the presence of Ca(2+) was characterized in single skinned fibres from rabbit psoas muscle. Pre-photolysis force was minimized using apyrase to remove contaminating ATP and ADP. After the initial force rise induced by ATP release, a rapid shortening ramp terminated by a step stretch to the original length was imposed, and the time course of the subsequent force redevelopment was again characterized. Force development after ATP release was accurately described by a lag phase followed by one or two exponential components. At 20 degrees C, the lag was 5.6 +/- 0.4 ms (s.e.m., n = 11), and the force rise was well fitted by a single exponential with rate constant 71 +/- 4 s(-1). Force redevelopment after shortening-restretch began from about half the plateau force level, and its single-exponential rate constant was 68 +/- 3 s(-1), very similar to that following ATP release. When fibres were activated by the addition of Ca(2+) in ATP-containing solution, force developed more slowly, and the rate constant for force redevelopment following shortening-restretch reached a maximum value of 38 +/- 4 s(-1) (n = 6) after about 6 s of activation. This lower value may be associated with progressive sarcomere disorder at elevated temperature. Force development following ATP release was much slower at 5 degrees C than at 20 degrees C. The rate constant of a single-exponential fit to the force rise was 4.3 +/- 0.4 s(-1) (n = 22), and this was again similar to that after shortening-restretch in the same activation at this temperature, 3.8 +/- 0.2 s(-1). We conclude that force development after ATP release and shortening-restretch are controlled by the same steps in the actin-myosin ATPase cycle. The present results and much previous work on mechanical-chemical coupling in muscle can be explained by a kinetic scheme in which force is generated by a rapid conformational change bracketed by two biochemical steps with similar rate constants -- ATP hydrolysis and the release of inorganic phosphate -- both of which combine to control the rate of force development.

Different Myofilament Nearest-Neighbor Interactions Have Distinctive Effects on Contractile Behavior

Cooperativity in contractile behavior of myofilament systems almost assuredly arises because of interactions between neighboring sites. These interactions may be of different kinds. Tropomyosin thin-filament regulatory units may have neighbors in steric blocking positions ( off) or steric permissive positions ( on). The position of these neighbors influence the tendency for the regulatory unit to assume the on or off state. Likewise, the tendency of a myosin cross-bridge to achieve a force-bearing state may be influenced by whether neighboring cross-bridges are in force-bearing states. Also, a cross-bridge in the force-bearing state may influence the tendency of a regulatory unit to enter the on state. We used a mathematical model to examine the influence of each of these three kinds of neighbor interactions on the steady-state force-pCa relation and on the dynamic force redevelopment process. Each neighbor interaction was unique in its effects on maximal Ca 2+-activated force, position, and symmetry of the force-pCa curve and on the Hill coefficient. Also, each neighbor interaction had a distinctive effect on the time course of force development as assessed by its rate coefficient, k dev. These diverse effects suggest that variations in all three kinds of nearest-neighbor interactions may be responsible for a wide variety of currently unexplained observations of myofilament contractile behavior.

Dynein-ADP as a Force-Generating Intermediate Revealed by a Rapid Reactivation of Flagellar Axoneme

Fragmented flagellar axonemes of sand dollar spermatozoa were reactivated by rapid photolysis of caged ATP. After a time lag of 10 ms, axonemes treated with protease started sliding disintegration. Axonemes without protease digestion started nanometer-scale high-frequency oscillation after a similar time lag. Force development in the sliding disintegration was measured with a flexible glass needle and its time course was corresponded well to that of the dynein-ADP intermediate production estimated using kinetic rates previously reported. However, with a high concentration (∼80 μM) of vanadate, which binds to the dynein-ADP intermediate and forms a stable complex of dynein-ADP-vanadate, the time course of force development in sliding disintegration was not affected at all. In the case of high frequency oscillation, the time lag to start the oscillation, the initial amplitude, and the initial frequency were not affected by vanadate, though the oscillation once started was damped more quickly at higher concentrations of vanadate. These results suggest that during the initial turnover of ATP hydrolysis, force generation of dynein is not blocked by vanadate. A vanadate-insensitive dynein-ADP is postulated as a force-generating intermediate.

Triiodothyronine reverses depressed contractile performance after excessive catecholamine stimulation

Background. Conflicting results have been reported regarding the acute effects of triiodothyronine (T 3) on myocardial contractile performance. The present study analyzes the role of T 3 in reversing the depressant effect of excessive catecholamine stimulation in isolated porcine left ventricular myocardium. Methods. Thirty-six left ventricular trabeculae (0.4 × 6.0 mm) obtained from 6 pigs were used for measurements of isometric force development, isotonic shortening, and intracellular calcium in three experimental series (measurement conditions: 37°C; optimal length; supramaximal electrical stimulation, 1 Hz; calcium measurement, fura-2 ratio method; frequency, 225 Hz). In series 1, isometric force development was measured before and after a 60-minute incubation with 10 −7 mol/L epinephrine in preparations with (n = 6) and without (n = 6) preceding fura-2 loading for calcium measurements. In series 2, the acute effects of a 30-minute administration of T 3 (10 −9 mol/L) on isometric force and intracellular calcium were analyzed (n = 6). In series 3, after simultaneous fura-2 loading and a 6-hour 10 −7 mol/L epinephrine exposure the effects of T 3 (10 −9 mol/L, 30 minutes) on force development, shortening, and intracellular calcium transient were analyzed. Results. Long-term and high-dose epinephrine exposure induced a severe contractile depression with a significant reduction of isometric force development ( p < 0.05) and increased diastolic ( p < 0.001) and systolic calcium ( p < 0.001). In normal porcine myocardium T 3 had no effect on the extent of isometric force generation but accelerated the time course of force development ( p < 0.05) and increased the calcium transient ( p < 0.001). After induction of myocardial depression by epinephrine exposure T 3 accelerated the intracellular calcium transients and reduced diastolic calcium. Triiodothyronine increased the shortening amplitude and the force amplitude ( p < 0.01). Conclusions. Triiodothyronine reverses depressed contractile performance after preceding high-dose epinephrine exposure in isolated porcine myocardium. Increased force amplitudes and unaltered or even reduced intracellular calcium transients argue in favor of a resensitization of the contractile apparatus for calcium by T 3. The study supports a potential role for T 3 treatment in depressed myocardium after previous excessive catecholamine exposure (eg, brain death, catecholamine treatment, ischemia).

Targeted ablation of the phospholamban gene is associated with a marked decrease in sensitivity in aortic smooth muscle.

Phospholamban (PLB) is a protein associated with the Ca(2+)-ATPase of the sarcoplasmic reticulum (SR) in cardiac, slow-twitch skeletal, and smooth muscle. PLB inhibits the SR Ca(2+)-ATPase in cardiac muscle; this inhibition is relieved on phosphorylation. The role of PLB in smooth muscle contractility is less clear. To elucidate the role of PLB in vascular smooth muscle contractility in vivo, we used a model in which the PLB gene was targeted in murine embryonic stem cells, generating mice deficient in PLB (PLB-). The PLB- mice exhibited no gross developmental abnormalities, but marked changes in aortic contractility were observed. The time course of force development with phenylephrine stimulation was faster in the PLB- aorta, suggesting changes in SR Ca2+ release. No differences were observed for KCl contractures between tissue types for either maximum forces observed or time course of force production; relaxation was faster in 7 of 11 arteries, but this trend did not attain statistical significance. The cumulative concentration-isometric force relations for the PLB- aorta were to the right of the wild-type for both KCl and phenylephrine stimulation, indicating a less sensitive tissue. To investigate whether the observed changes were related to SR function, we inhibited the SR Ca(2+)-ATPase with cyclopiazonic acid (CPA). CPA treatment resulted in a leftward shift of the concentration-isometric force relations for both aorta types, as expected after removal of a major Ca2+ uptake system. Most interestingly, the differences between PLB and wild-type aorta were abolished by SR inhibition. Our results suggest that PLB is a regulator of the SR Ca2+ pump in mouse aorta and plays a regulatory role in both KCl-induced and receptor-mediated contractility in vascular smooth muscle.

Mechanical response to photolytic ATP pulses of skinned muscle fibres pre-activated with a small pulse of ATP.

(1) Skinned fibres from rat psoas muscle were placed in oil and activated at 10 degrees C by pulse photolysis of caged ATP. From the force and stiffness transients on small ATP pulses, we estimated the concentration and rate of ATP hydrolysis of the crossbridges in the fibres. They were 0.14 mM and 0.16 mMs-1, respectively, and thus the turnover rate was 1.1 s-1. (2) To examine the mechanical properties of the physiological ADP-bound crossbridges, we took a double-pulse protocol: the skinned fibre was first activated with a conditioning ATP pulse (0.2-0.5 mM). Several (3-5) seconds later, when almost all the ATP should have been hydrolysed into ADP+P(i), we applied another ATP pulse (0.2-1 mM) to test the fibre. (3) An analysis of the transients on the test pulses indicated that the conditioning pulse slowed the rate of crossbridge detachment. The time course of force development was thus expected to be also delayed, but was not. (4) We suggested that the rigor crossbridge might differ from the nucleotide-free intermediate in the physiological reaction cycle.

Effects of ethylene glycol and calcium on the kinetics of contraction induced by photo-release of low concentrations of ATP in rat psoas muscle fibres.

To induce isometric contraction in the absence of Ca2+ (10 mM EGTA), low concentrations (130 microM) of ATP were photoreleased from caged ATP in skinned fibres from rat psoas muscle at 15-16 degrees C. The magnitude of contraction was independent of the concentration of EGTA (1-30 mM). Each isometric transient (i) was paired with another (s) obtained under the same conditions but with 0.4% muscle stretch to elevate the rigor force before photolysis. The algebraic difference (d) between i and s was assumed to represent detachment of the crossbridges. The time course of force development (f) by the reattached crossbridges could be estimated by subtracting an appropriately scaled d from i (or s). Ethylene glycol (20% in solvent) reduced the magnitude and the rate of rise of f, although it scarcely affected d, suggesting that ethylene glycol inhibited reattachment of the crossbridges but not their detachment. The presence of Ca2+ (50 microM) increased the magnitude of f, but did not affect its time course (130 microM ATP). Detachment, d, was not influenced by Ca2+ in terms of both extent and rate. The effect of Ca2+ in the presence of ethylene glycol was indistinguishable from that in its absence. Ethylene glycol did not seem to substantially affect the extent of Ca-regulation on the contractile activity.

Rapid increase in inositol phosphate levels in norepinephrine-stimulated vascular smooth muscle

We examined the correlation between agonist-stimulated increases in inositol phosphates and force development in vascular smooth muscle. Segments of rat tail artery were preincubated with [3H]inositol and treated with norepinephrine (10(-5) M) for 3-10 s. Tissue levels of inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) were measured. IP and IP2 increased significantly after 3 s of norepinephrine treatment. IP3 increased significantly after 5 s of norepinephrine treatment. Analysis of tissue extracts by high-pressure liquid chromatography demonstrated that the only isomer of IP3 present in any tissue extract was the 1,4,5-isomer [Ins(1,4,5)P3]. Contractile response to norepinephrine stimulation showed that the increase in inositol phosphates coincides well with the time course of force development. This is the first report demonstrating such an early increase in Ins(1,4,5)P3 in agonist-stimulated vascular smooth muscle. These results are consistent with the hypothetical role of Ins(1,4,5)P3 as a mediator linking agonist-receptor activation to increased intracellular calcium and force development in norepinephrine-stimulated vascular smooth muscle.

Cognitive spatial-motor processes

The effects of an external constant force bias on the information transmitted (Ti) by the direction of isometric force exerted in 2-dimensional (2-D) space by human subjects were studied using an isometric manipulandum and random dot stereograms generated in a color display (Massey et al. 1988, Massey et al. 1990). Subjects exerted force on the manipulandum such that a visual force-feedback cursor would move in the direction of a visually defined stimulus in the stereo display. The time course of force development and the gain of directional information during increasing force intensity were also studied. We found the following. (a) When no bias force was applied, the force exerted by the subject increased from near zero to greater than 200 gram-force at the end of a trial and was close to the visually defined direction. When a constant bias force of 110 gram-force was applied in various directions in blocks of trials, the force exerted by the subject increased in time, as above; however, its direction also changed in time so that the instantaneous vector sum of the bias force and the force exerted by the subject pointed close to the visually defined direction. The Ti and the reaction time (RT) did not differ significantly in the two experimental conditions. These results suggest that the directional control of isometric forces is very efficient, especially in relation to visuomotor coordination. (b) The Ti was calculated at various levels of force intensity, as the latter increased from approximately 50 gram-force to 200 gram-force.(ABSTRACT TRUNCATED AT 250 WORDS)

Force measurements from voltage-clamped guinea pig ventricular myocytes

We describe the first observations of isolated mammalian guinea pig ventricular myocytes that combine measurements of contractile force with the voltage-clamp method. The myocytes were attached by poly-L-lysine to the beveled ends of a pair of thin glass rods having a compliance of 0.76 m/N. The contractile force of a cell caused a 1- to 3-microm displacement of the rods; the motion of which was converted to an output voltage by phototransistors. By the use of the whole cell patch-clamp technique, the cells were depolarized at 1 Hz with 200-ms-long clamp pulses from -45 to +5 mV (35 degrees C, 3.6 mM CaCl2). Isometric force began after a latency of 7 +/- 2 ms, peaked at 93 +/- 21 ms, and relaxed (90%) at 235 +/- 63 ms. The time course of force was always faster than that of isotonic shortening (time to peak 154 +/- 18 ms). With 400-ms-long depolarizations, a tonic component was recorded as either sustained force or sustained shortening that decayed on repolarization. Substitution of Ca by Sr in the bath increased the inward current through Ca channels but slowed down the time course of force development. The results are consistent with the hypothesis that activator calcium derives mainly from internal stores and that Ca release needs Ca entry through channels.

Kinetics of contraction initiated by flash photolysis of caged adenosine triphosphate in tonic and phasic smooth muscles.

Laser flash photolysis of caged adenosine triphosphate (ATP), in the presence of Ca2+, was used to examine the time course of isometric force development from rigor states in glycerinated tonic (rabbit trachealis) and phasic (guinea-pig ileum and portal vein) smooth muscles. Photolytic liberation of ATP from caged ATP initiated force development, at 20 degrees C, with half-time (t1/2) of 5.4 s in trachealis and 1.2-2.2 s in the phasic muscles. Prior to photolysis, some muscles were phosphorylated with ATP plus okadaic acid (an inhibitor of myosin light-chain phosphatase) or thiophosphorylated with ATP gamma S to fully activate the regulatory system, before turning on the contractile apparatus. In these prephosphorylated muscles, force development, after caged ATP photolysis, was more rapid than in the unphosphorylated muscles, but the t1/2 values for trachealis (0.8-1.1 s) were still longer than for ileum and portal-vein muscles (0.20-0.25 s). The results suggest that both the contractile machinery and the regulatory system are slower in the tonic than in the phasic smooth muscles. The time course of force development for each muscle type was sigmoidal, with an initial delay (td) of approximately 10% of the t1/2 value. Some possible chemical and mechanical origins of the delay are discussed.

Mathematical model of cardiac mechanogram rhythmicity (based on mollusc heart).

1. The time course of force development by the heart is modelled by Gompertz kinetics from the product of two terms: a cumulative increase in relative number of activated "contractile units", and an exponential decrease in contractile force. 2. For each beat, an "initial condition" is specified by an "intrinsic tension" parameter, and a specific rate of change of tension; cardioactive agents change these specifications. 3. Depending on parameter values, heartbeats are predicted that are constant, or in which the frequency, amplitude and baseline tension are appropriate to inhibited or augmented cardiac activity.

Force development during sustained locomotion: a determinant of gait, speed and metabolic power.

This paper develops three simple ideas about force development during sustained locomotion which provide some insights into the mechanisms that determine why animals change gait, how fast they can run, and how much metabolic energy they consume. The first idea is that the alternate stretch-shorten pattern of activity of the muscles involved in locomotion allows muscle-tendon units to function as springs, affecting the amount of force a given cross-sectional area of muscle develops, and the metabolic requirements of the muscles for force development. Animals select speeds and stride frequencies which optimize the performance of these springs. The second idea is that muscle stress (force/cross-sectional area) determines when animals change gait, how fast they run and their peak accelerations and decelerations. It is proposed that terrestrial birds and mammals develop similar muscle stresses under equivalent conditions (i.e. preferred speed within a gait) and that animals change gaits in order to reduce peak stresses as they increase speed. Finally, evidence is presented to support the idea that it is the time course of force development during locomotion, rather than the mechanical work that the muscles perform, that determines the metabolic cost of locomotion.