Biomedical subjects

L E Ford

Publications and source records attributed to L E Ford.

At least 19 recordsLinked to original sources

PubMed2002

Myosin light chain phosphorylation facilitates in vivo myosin filament reassembly after mechanical perturbation.

Phosphorylation of the 20-kDa regulatory myosin light chain (MLC) of smooth muscle is known to cause monomeric myosins in solution to self-assemble into thick filaments. The role of MLC phosphorylation in thick filament formation in intact muscle, however, is not clear. It is not known whether the phosphorylation is necessary to initiate thick filament assembly in vivo. Here we show, by using a potent inhibitor of MLC kinase (wortmannin), that the MLC phosphorylation and isometric force in trachealis muscle could be abolished without affecting calcium transients. By measuring cross-sectional densities of the thick filaments electron microscopically, we also show that inhibition of MLC phosphorylation alone did not cause disassembly of the filaments. The unphosphorylated thick filaments, however, partially dissolved when the muscle was subjected to oscillatory strains (which caused a 25% decrease in the thick filament density). The postoscillation filament density recovered to the preoscillation level only when wortmannin was removed and the muscle was stimulated. The data suggest that in vivo thick filament reassembly after mechanical perturbation is facilitated by the cyclic MLC phosphorylation associated with repeated stimulation.

Androstadienes↗

PubMed2001

Effects of substituting uridine triphosphate for ATP on the crossbridge cycle of rabbit muscle.

1. Substituting uridine triphosphate (UTP) for ATP as a substrate for rabbit skeletal myosin and actin at 4 degrees C slowed the dissociation of myosin-S1 from actin by threefold, and hydrolysis of the nucleotide by sevenfold, without a decrease in the rates of phosphate or uridine diphosphate dissociation from actomyosin. 2. The same substitution in skinned rabbit psoas fibres at 2-3 degrees C reduced the maximum shortening velocity by 56 % and increased the force asymptote of the force-velocity curve relative to force (alpha/P(o)) by 112 % without altering the velocity asymptote, beta. It also decreased isometric force by 35 % and isometric stiffness by 20 %, so that the stiffness/force ratio was increased by 23 %. 3. Tension transient experiments showed that the stiffness/force increase was associated with a 10 % reduction in the amplitude of the rapid, partial (phase 2) recovery relative to the isometric force, and the addition of two new components, one that recovered at a step-size-independent rate of 100 s(-1) and another that did not recover following the length change. 4. The increased alpha/P(o) with constant beta suggests an internal load, as expected of attached crossbridges detained in their movement. An increased stiffness/force ratio suggests a greater fraction of attached bridges in low-force states, as expected of bridges with unhydrolyzed UTP detained in low-force states. Decreased phase 2 recovery suggests the detention of high-force bridges, as expected of slowed actomyosin dissociation by nucleotide. 5. These results suggest that the separation of hydrolysed phosphates from nucleotides occurs early in the attached phase of the crossbridge cycle, near and possibly identical to a transition to a firmly attached, low-force state from an initial state where bridges with hydrolysed nucleotides are easily detached by shortening.

Actins↗

PubMed2001

Myosin thick filament lability induced by mechanical strain in airway smooth muscle.

Airway smooth muscle adapts to different lengths with functional changes that suggest plastic alterations in the filament lattice. To look for structural changes that might be associated with this plasticity, we studied the relationship between isometric force generation and myosin thick filament density in cell cross sections, measured by electron microscope, after length oscillations applied to the relaxed porcine trachealis muscle. Muscles were stimulated regularly for 12 s every 5 min. Between two stimulations, the muscles were submitted to repeated passive +/- 30% length changes. This caused tetanic force and thick-filament density to fall by 21 and 27%, respectively. However, in subsequent tetani, both force and filament density recovered to preoscillation levels. These findings indicate that thick filaments in airway smooth muscle are labile, depolymerization of the myosin filaments can be induced by mechanical strain, and repolymerization of the thick filaments underlies force recovery after the oscillation. This thick-filament lability would greatly facilitate plastic changes of lattice length and explain why airway smooth muscle is able to function over a large length range.

Animals↗

PubMed2001

Simple freezing apparatus for resolving rapid metabolic events associated with smooth muscle activation.

A method is described for freezing thin strips of smooth muscle by replacing physiological saline in the muscle chamber with cold organic solvent in <100 ms. Calculations suggest that, with a perfectly stirred boundary at the tissue surface, freezing could occur within approximately 15 ms at the center of a 200-microm-thick piece of tissue by use of acetone coolant at -78.5 degrees C and in approximately half the time with either isopentane at its freezing point (-160 degrees C) or aluminum chilled with liquid nitrogen. Myosin light chain phosphorylation in muscles frozen with cold acetone began to rise approximately 200 ms earlier than force and increased at a much more rapid rate. The difference in onsets of the two processes reflects the delay in arresting phosphorylation plus two lags associated with force generation, attachment of phosphorylated bridges followed by force generating movements of the attached bridges. The much more rapid rise of phosphorylation, once it began, suggests that most of this delay is due to physiological lags and not to slow arrest of metabolism.

Animals↗

PubMed2001

Relationship between myosin phosphorylation and contractile capability of canine airway smooth muscle.

To better understand excitation-contraction coupling in smooth muscle, myosin phosphorylation and force-velocity properties of canine tracheal muscle were compared during the rise and early plateau of force in electrically stimulated tetani. Velocity reached a peak of approximately 1.5 times plateau value when force had risen to approximately 45% of its maximum value and then declined progressively. Except early in the tetanus, when phosphorylation rose rapidly, maximum power and phosphorylation had nearly parallel time courses, reaching peaks of 1.2-1.3 times reference at 6-8 s before declining to the plateau level at approximately 12 s. Force, velocity, maximum power, and phosphorylation fell somewhat during the plateau, with the closest correlation between phosphorylation and power. These results suggest that 1) early velocity slowing is not associated with light chain dephosphorylation and 2) maximum power, which we use to signal changes in activation, is closely correlated with the degree of light chain phosphorylation, at least when phosphorylation level is not changing rapidly. Dissociation of these two properties would be expected early in the tetanus if phosphorylation precedes mechanical activity.

Animals↗

PubMed2000

Versatile, high-speed force transducer using a laser diode beam as an optical lever.

A force transducer with variable sensitivity and speed is described. Its moving element is a cantilever beam that projects vertically into a muscle bath. A brace constrains bending of the beam to a short, proximal "hinge." Rotation of the beam about the hinge is amplified 30-fold by an optical lever consisting of a laser diode beam reflected from a mirror on the cantilever to a photodiode pair. This design places the electrical components at a distance from the damp environment of the muscle bath. Large changes in sensitivity and speed can be obtained by substituting different cantilevers. Smaller changes can be made by varying the length of the hinge. A transducer with a 6-mm cantilever optimized for the study of single, skinned skeletal muscle fibers is described in detail. This device had a resonant frequency of 22 kHz and sensitivity such that the total root-mean-square noise in the circuit was more than 500-fold smaller than the expected maximum force. Variations of this device with orders of magnitude different sensitivities are also described.

Amplifiers, Electronic↗

PubMed2000

Gender- and height-related limits of muscle strength in world weightlifting champions.

To assess factors that limit human muscle strength and growth, we examined the relationship between performance and body dimensions in the world weightlifting champions of 1993-1997. Weight lifted varied almost exactly with height squared (Ht(2.16)), suggesting that muscle mass scaled almost exactly with height cubed (Ht(3.16)) and that muscle cross-sectional area was closely correlated with body height, possibly because height and the numbers of muscle fibers in cross section are determined by a common factor during maturation. Further height limitations of muscle strength were shown by only one male champion >/=183 cm and no female champions >/=175 cm. The ratio of weight lifted to mean body cross-sectional area was approximately constant for body-weight classes </=83 kg for men and </=64 kg for women and decreased abruptly for higher weight classes. These findings suggest a nearly constant fraction of body mass devoted to muscle in lighter lifters and a lesser fraction in heavier lifters. Analysis also suggests that contractile tissue comprises approximately 30% less body mass in female champions.

Body Height↗

PubMed2000

Series-to-parallel transition in the filament lattice of airway smooth muscle.

Force-velocity curves measured at different times during tetani of sheep trachealis muscle were analyzed to assess whether velocity slowing could be explained by thick-filament lengthening. Such lengthening increases force by placing more cross bridges in parallel on longer filaments and decreases velocity by reducing the number of filaments spanning muscle length. From 2 s after the onset of stimulation, when force had achieved 42% of it final value, to 28 s, when force had been at its tetanic plateau for approximately 15 s, velocity decreases were exactly matched by force increases when force was adjusted for changes in activation, as assessed from the maximum power value in the force-velocity curves. A twofold change in velocity could be quantitatively explained by a series-to-parallel change in the filament lattice without any need to postulate a change in cross-bridge cycling rate.

Actin Cytoskeleton↗

PubMed1997

Detachment of low-force bridges contributes to the rapid tension transients of skinned rabbit skeletal muscle fibres.

1. To probe the cross-bridge cycle and to learn more about the cardioplegic agent BDM (2,3-butanedione monoxime), its effects on the force-velocity properties and tension transients of skinned rabbit muscle fibres were studied at 1-2 degrees C and pH 7.0. 2. Three millimolar BDM decreased isometric force by 50%, velocity by 29%, maximum power by 73%, and stiffness by 25%, so that the relative stiffness (stiffness/force ratio) increased by 50% compared with reference conditions in the absence of BDM. 3. Tension transients obtained under the reference condition (0 BDM) could be represented by three components whose instantaneous stiffness accounted for the initial (Phase 1) force deviation and whose exponential recoveries caused the rapid, partial (Phase 2) force recovery following the step. The fastest component had non-linear extension-force properties that accounted for about half the isometric stiffness and it recovered fully. The two slower components had linear extension-force properties that together accounted for the other half of the sarcomere stiffness. These components recovered only partially following the step, producing the intermediate (T2) level which the force approached during Phase 2. 4. Matching the force transients obtained under test conditions (3 mM BDM) required three alterations: (1) reducing the amplitude of the two slower components by 50%, in proportion to isometric force, (2) adding a non-relaxing component and (3) decreasing the amplitude of the rapidly recovering component by 12.5% so that its relative amplitude (amplitude/isometric force) was increased by 75%. The non-recovering component and the increase in relative amplitude of the rapid component were responsible for the increase in relative stiffness of the fibres produced by BDM. The rapidly recovering component had the same time constant and step-size-dependent recovery rates as the fastest of the three mono-exponential components isolated from the tension transient response under the reference condition. BDM therefore appeared to augment the fastest component of the tension transient under the reference condition. 5. The results suggest that BDM detains cross-bridges in low-force, attached states. Since these bridges are attached, they contribute to sarcomere stiffness. Since they are detained, relaxation or reversal of their immediate responses is probably due to bridge detachment rather than to their undergoing the power stroke. The observation that a portion of the test response matched the fastest component of the reference response when the amplitude of the fastest component was increased suggests that a part of the normal rapid, transient tension recovery following a release step is due to detachment of low-force bridges moved to negative-force positions by the step.

Animals↗

PubMed1997

Exchange of ATP for ADP on high-force cross-bridges of skinned rabbit muscle fibers.

The contractile properties of rabbit skinned muscle fibers were studied at 1-2 degrees C in different concentrations of MgATP and MgADP. Double-reciprocal plots of maximum velocity against MgATP concentration at different MgADP concentrations all extrapolated to the same value. This finding suggests that MgATP and MgADP compete for the same site on the cross-bridge, and that the exchange of MgATP for MgADP occurs without a detectable step intervening. The K(m) for ATP was 0.32 mM. The K(i) for MgADP was 0.33 mM. Control experiments suggested that the tortuosity of diffusion paths within the fibers reduced the radial diffusion coefficients for reactants about sixfold. Increasing MgADP from 0.18 to 2 mM at 5 mM ATP or lowering MgATP from 10 to 2 mM at 0.18 mM MgADP, respectively, increased isometric force by 25% and 23%, increased stiffness by 10% and 20%, and decreased maximum velocity by 35% and 31%. Two mechanisms appeared to be responsible. One detained bridges in high-force states, where they recovered from a length step with a slower time course. The other increased the fraction of attached bridges without altering the kinetics of their responses, possibly by an increased activation resulting from cooperative effects of the detained, high-force bridges. The rigor bridge was more effective than the ADP-bound bridge in increasing the number of attached bridges with unaltered kinetics.

Adenosine Diphosphate↗

PubMed1995

Plasticity in canine airway smooth muscle.

The large volume changes of some hollow viscera require a greater length range for the smooth muscle of their walls than can be accommodated by a fixed array of sliding filaments. A possible explanation is that smooth muscles adapt to length changes by forming variable numbers of contractile units in series. To test for such plasticity we examined the muscle length dependence of shortening velocity and compliance, both of which will vary directly with the number of thick filaments in series. Dog tracheal smooth muscle was studied because its cells are arrayed in long, straight, parallel bundles that span the length of the preparation. In experiments where muscle length was changed, both compliance and velocity showed a strong dependence on muscle length, varying by 1.7-fold and 2.2-fold, respectively, over a threefold range of length. The variation in isometric force was substantially less, ranging from a 1.2- to 1.3-fold in two series of experiments where length was varied by twofold to an insignificant 4% variation in a third series where a threefold length range was studied. Tetanic force was below its steady level after both stretches and releases, and increased to a steady level with 5-6 tetani at 5 min intervals. These results suggest strongly that the number of contractile units in series varies directly with the adapted muscle length. Temporary force depression after a length change would occur if the change transiently moved the filaments from their optimum overlap. The relative length independence of the adapted force is explained by the reforming of the filament lattice to produce optimum force development, with commensurate changes of velocity and compliance.

Adaptation, Physiological↗

PubMed1994

A program for developing a comprehensive mathematical description of the crossbridge cycle of muscle.

We describe a computer modeling system for determining the changes of force, fraction of attached crossbridges, and crossbridge flux rate through a specifiable transition in response to length changes imposed on a crossbridge model of muscle. The crossbridge cycle is divided into multiple attached and detached states. The rates of transition from one state to another are defined by rate coefficients that can either be constant or vary with the position of the crossbridge relative to the thin-filament attachment site. This scheme leads to a system of differential equations defining the rates of change for the fractions of bridges in each state. Solutions for this system of equations are obtained at specified times during and after a length change using a method for systems with widely varying time constants (C. W. Gear, 1971, Numerical Initial Value Problems in Ordinary Differential Equations, Prentice-Hall, Englewood Cliffs, NJ). Crossbridges are divided into discrete populations that differ both in their axial displacement with respect to thin filament attachment sites and with respect to the twist of the actin helix. Separate solutions are made for the individual populations and are then averaged to obtain the ensemble response. Force is determined as the sum of the product of the force associated with each state multiplied by the fraction of bridges in that state. A measure of metabolic rate is determined as the net flux through one of the crossbridge transitions. When the force-extension characteristics of the individual crossbridges are linear and the filaments are noncompliant the fraction of attached bridges is equivalent to sarcomere stiffness. To illustrate the operation of the program, we also describe here some results obtained with a simplified scheme.

Actins↗

PubMed1994

Plasticity in smooth muscle, a hypothesis.

The controversial finding that the thick filaments of smooth muscle can be evanescent leads to the hypothesis that the large functional range of this muscle is accommodated by plastic rearrangements that place more thick filaments in series at longer lengths. Our preliminary finding that the shortening velocity and compliance of dog tracheal muscle were strongly dependent on adapted muscle length, while force was much less length dependent, supports this hypothesis (V.R. Pratusevich, C.Y. Seow, and L.E. Ford. Biophys. J. 66: A139, 1994). The hypothesis leads to two further corollaries. The first is that the lengthening of the thick filaments that must accompany their reformation will cause a series to parallel transition: fewer long filaments span the muscle length, but the longer filaments have more cross bridges acting in parallel. The second is that there is more than one activating mechanism in smooth muscle. It is known that myosin light chain phosphorylation activates the actomyosin ATPase, but this same phosphorylation also causes a structural change that facilitates filament formation. The consideration that the unaggregated, phosphorylated myosin must be prevented from competing with myosin in thick filaments and hydrolyzing ATP suggests that there must be a second mechanism that must allow the thin filaments to interact selectively with filamentous myosin. This need for a second activating mechanism may explain the presence of tropomyosin, calponin, and caldesmon on thin filaments. Although the two corollaries follow from the initial hypothesis, it should be emphasized that the three are not mutually dependent, and that the proof or disproof of any one of them would not prove or disprove the others.

Animals↗

PubMed1994

Valve resistance.

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Heart Valves↗

PubMed1993

Myocardial energetics.

Inotropic alterations may alter contractile efficiency. Positive inotropic agents act mainly, if not exclusively, by increasing activation, which has very little effect on contractile efficiency. The metabolic effects of hypoxia may depress the contractile machinery directly. Acidosis is likely to decrease efficiency while an elevated phosphate, by itself, is likely to have little effect on efficiency.

Animals↗

PubMed1993

High ionic strength and low pH detain activated skinned rabbit skeletal muscle crossbridges in a low force state.

The effects of varying pH and ionic strength on the force-velocity relations and tension transients of skinned rabbit skeletal muscle were studied at 1-2 degrees C. Both decreasing pH from 7.35 to 6.35 and raising ionic strength from 125 to 360 mM reduced isometric force by about half and decreased sarcomere stiffness by about one-fourth, so that the stiffness/force ratio was increased by half. Lowering pH also decreased maximum shortening velocity by approximately 29%, while increasing ionic strength had little effect on velocity. These effects on velocity were correlated with asymmetrical effects on stiffness. The increase in the stiffness/force ratio with both interventions was manifest as a greater relative force change associated with a sarcomere length step. This force difference persisted for a variable time after the step. At the high ionic strength the force difference was long-lasting after stretches but relaxed quickly after releases, suggesting that the structures responsible would not impose much resistance to steady-state shortening. The opposite was found in the low pH experiments. The force difference relaxed quickly after stretches but persisted for a long time after releases. Furthermore, this force difference reached a constant value of approximately 8% of isometric force with intermediate sizes of release, and was not increased with larger releases. This value was almost identical to the value of an internal load that would be sufficient to account for the reduction in maximum velocity seen at the low pH. The results are interpreted as showing that both low pH and high ionic strength inhibit the movement of crossbridges into the force-generating parts of their cycle after they have attached to the actin filaments, with very few other effects on the cycle. The two interventions are different, however, in that detained bridges can be detached readily by shortening when the detention is caused by high ionic strength but not when it is caused by low pH.

Actins↗

PubMed1992

Contribution of damped passive recoil to the measured shortening velocity of skinned rabbit and sheep muscle fibres.

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 greater than 0.1). Maximum overall muscle velocity measured from the slack test was significantly (p greater than 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.

Animals↗

PubMed1992

Changes in valvular resistance, power dissipation and myocardial reserve with aortic valvuloplasty.

Balloon aortic valvuloplasty results in small changes in valve area with great symptomatic improvement in some patients, while others have little relief with greater increases in valve area. Alternative indices to valve area may help explain this clinical discrepancy. A calculation of valve area does not provide a means of assessing the load imposed by a stenotic valve, while the complementary index valve resistance, defined as the quotient of mean pressure difference divided by flow, allows many other hemodynamic calculations and may provide an additional measure of the hemodynamic importance of valvular obstructions. To assess the value of these calculations, we studied hemodynamic changes in thirty elderly patients undergoing valvuloplasty for aortic stenosis. The valve area, as calculated by the Gorlin formula, increased by an average 67% (0.59 cm2 to 0.95 cm2), while hemodynamic resistance decreased by an average 52% (453 to 207 dyne.sec.cm5). The values of resistance were used to predict pressure gradients and work loads at different cardiac outputs. The increase in myocardial reserve with valvuloplasty was calculated as the increase in cardiac output that could be achieved at the pre-valvuloplasty value of either total ventricular pressure or ventricular work. These calculations assumed that valvular resistance did not change with cardiac output and that peripheral resistance varied inversely to cardiac output so as to maintain a constant aortic (systemic) pressure. The increase in myocardial reserve was 18% when ventricular work rate was the limiting factor, and 103% when pressure was limiting. The increase in reserve may be closer to the higher value since the myocardial work rate is probably not limited by myocardial energy in the absence of coronary artery disease. Four patients who did not do well clinically were characterized by small increases in reserve, either because of inadequate dilatation of the valve or because the original stenosis was not severe. Valve resistance, myocardial reserve, and ventricular work may be calculated using standard hemodynamic measurements. In conjunction with aortic valve area, these indices provide significant complimentary information and may further elucidate the hemodynamic consequences of valvular obstruction.

Aged↗