Rabbit Muscle Fibers Research Articles

Detection of Super-Relaxed Myosin in Specific Human Skeletal Muscle Fiber Types

We are studying the super-relaxed state of myosin, with the goal of developing novel therapeutic tools for treatment of contractile and metabolic disorders. In muscle, myosins use the energy from ATP hydrolysis to perform mechanical work during muscle contraction, and to help maintain basal metabolic rate at rest. The myosin super-relaxed state (SRX) in skeletal muscle is hypothesized to play an important role in regulating muscle cell contractility and thermogenesis, yet its physiological role in skeletal muscle remains elusive. The SRX is a biochemical state of myosin with slowed ATP turnover kinetics, due to auto-inhibition of the catalytic domains. Previous work on myosin SRX focused on samples obtained from rabbits and mice. Here, we have used refined quantitative epifluorescence microscopy of fluorescent MANT-ATP to measure single-nucleotide turnover of myosin in skinned skeletal muscle fibers from human donors for the first time. As with most mammalian muscles, the human vastus lateralis is composed of fibers expressing different myosin heavy chain (MHC) isoforms (I, IIA and IIX), which was determined for each fiber tested using gel electrophoresis. Preliminary results show that human skeletal SRX from MHC I fibers have faster ATP turnover compared to MHC II fibers, as previously observed in rabbit muscle fibers. From this work, we are making significant progress toward understanding the biochemical and physiological significance of myosin SRX in human skeletal muscle.

Tropomyosin movement is described by a quantitative high-resolution model of X-ray diffraction of contracting muscle.

Contraction of skeletal and cardiac muscle is controlled by Ca2+ ions via regulatory proteins, troponin (Tn) and tropomyosin (Tpm) associated with the thin actin filaments in sarcomeres. In the absence of Ca2+, Tn-C binds actin and shifts the Tpm strand to a position where it blocks myosin binding to actin, keeping muscle relaxed. According to the three-state model (McKillop and Geeves Biophys J 65:693-701, 1993), upon Ca2+ binding to Tn, Tpm rotates about the filament axis to a 'closed state' where some myosin heads can bind actin. Upon strong binding of myosin heads to actin, Tpm rotates further to an 'open' position where neighboring actin monomers also become available for myosin binding. Azimuthal Tpm movement in contracting muscle is detected by low-angle X-ray diffraction. Here we used high-resolution models of actin-Tpm filaments based on recent cryo-EM data for calculating changes in the intensities of X-ray diffraction reflections of muscle upon transitions between different states of the regulatory system. Calculated intensities of actin layer lines provide a much-improved fit to the experimental data obtained from rabbit muscle fibers in relaxed and rigor states than previous lower-resolution models. We show that the intensity of the second actin layer line at reciprocal radii from 0.15 to 0.3nm-1 quantitatively reports the transition between different states of the regulatory system independently of the number of myosin heads bound to actin.

Comparison of force and shortening velocity of fast and slow rabbit muscle fibers at different temperatures

The temperature dependence of force, maximal shortening velocity and power of maximally activated single permeabilized fibers from fast and slow muscles of the rabbit were recorded in a temperature range from 10 to 35 degrees C with 5 degrees C step. It was found that temperature dependence of force of both types of fibers is identical. Averaged maximal shortening velocity in the slow fibers, unlike the fast fibers, had no statistically significant temperature dependence that is not in agreement with the data obtained on intact rat muscle fibers and in an in vitro motility assay. However maximal shortening velocity in each individual slow fiber did depend on temperature. The temperature dependence of power of the slow fibers was lower than that of the fast ones. Because of large data scattering the average temperature dependence of power of the slow fibers was significantly lower than that in individual slow fibers.

The Fraction of Myosin Motors That Participate in Isometric Contraction of Rabbit Muscle Fibers at Near-Physiological Temperature

The duty ratio, or the part of the working cycle in which a myosin molecule is strongly attached to actin, determines motor processivity and is required to evaluate the force generated by each molecule. In muscle, it is equal to the fraction of myosin heads that are strongly, or stereospecifically, bound to the thin filaments. Estimates of this fraction during isometric contraction based on stiffness measurements or the intensities of the equatorial or meridional x-ray reflections vary significantly. Here, we determined this value using the intensity of the first actin layer line, A1, in the low-angle x-ray diffraction patterns of permeable fibers from rabbit skeletal muscle. We calibrated the A1 intensity by considering that the intensity in the relaxed and rigor states corresponds to 0% and 100% of myosin heads bound to actin, respectively. The fibers maximally activated with Ca2+ at 4°C were heated to 31–34°C with a Joule temperature jump (T-jump). Rigor and relaxed-state measurements were obtained on the same fibers. The intensity of the inner part of A1 during isometric contraction compared with that in rigor corresponds to 41–43% stereospecifically bound myosin heads at near-physiological temperature, or an average force produced by a head of ∼6.3 pN.

Temperature jump induced force generation in rabbit muscle fibres gets faster with shortening and shows a biphasic dependence on velocity.

We examined the tension responses to ramp shortening and rapid temperature jump (<0.2 ms, 3-4 degrees C T-jump) in maximally Ca(2+)-activated rabbit psoas muscle fibres at 8-9 degrees C (the fibre length (L(0)) was approximately 1.5 mm and sarcomere length 2.5 microm). The aim was to investigate the strain sensitivity of crossbridge force generation in muscle. The T-jump induced tension rise was examined during steady shortening over a wide range of velocities (V) approaching the V(max) (V range approximately 0.01 to approximately 1.5 L(0) s(1)). In the isometric state, a T-jump induced a biphasic tension rise consisting of a fast (approximately 50 s(1), phase 2b) and a slow (approximately 10 s(1), phase 3) component, but if treated as monophasic the rate was approximately 20 s(1). During steady shortening the T-jump tension rise was monophasic; the rate of tension rise increased linearly with shortening velocity, and near V(max) it was approximately 200 s(1), approximately 10x faster than in the isometric state. Relative to the tension reached after the T-jump, the amplitude increased with shortening velocity, and near V(max) it was 4x larger than in the isometric state. Thus, the temperature sensitivity of muscle force is markedly increased with velocity during steady shortening, as found in steady state experiments. The rate of tension decline during ramp shortening also increased markedly with increase of velocity. The absolute amplitude of T-jump tension rise was larger than that in the isometric state at the low velocities (<0.5 L(0) s(1)) but decreased to below that of the isometric state at the higher velocities. Such a biphasic velocity dependence of the absolute amplitude of T-jump tension rise implies interplay between, at least, two processes that have opposing effects on the tension output as the shortening velocity is increased, probably enhancement of crossbridge force generation and faster (post-stroke) crossbridge detachment by negative strain. Overall, our results show that T-jump force generation is strain sensitive and becomes considerably faster when exposed to negative strain. Thus the crossbridge force generation step in muscle is both temperature sensitive (endothermic) and strain sensitive.

Single-Molecule Measurement of the Stiffness of the Rigor Myosin Head

The force-extension curve of single myosin subfragment-1 molecules, interacting in the rigor state with an actin filament, has been investigated at low [ATP] by applying a slow triangle-wave movement to the optical traps holding a bead-actin-bead dumbbell. In combination with a measurement of the overall stiffness of the dumbbell, this allowed characterization of the three extensible elements, the actin-bead links and the myosin. Simultaneously, another method, based on an analysis of bead position covariance, gave satisfactory agreement. The mean covariance-based estimate for the myosin stiffness was 1.79 pN/nm (SD = 0.7 pN/nm; SE = 0.06 pN/nm ( n = 166 myosin molecules)), consistent with a recent report (1.7 pN/nm) from rabbit muscle fibers. In the triangle-wave protocol, the motion of the trapped beads during interactions was linear within experimental error over the physiological range of force applied to myosin (±10 pN), consistent with a Hookean model; any nonlinear terms could not be characterized. Bound states subjected to forces that resisted the working stroke (i.e., positive forces) detached at a significantly lower force than when subjected to negative forces, which is indicative of a strain-dependent dissociation rate.

Reply to Barclay and Loiselle

reply: We appreciate the interest expressed by Drs. Barclay and Loiselle ([2][1]) in our recent paper ([11][2]). We used N -benzyl- p -toluene sulfonamide (BTS) to inhibit cross-bridge force production and myosin ATPase-dependent ATP splitting (measured with analytical biochemistry). BTS has been

Direct Modeling of X-Ray Diffraction Pattern from Skeletal Muscle in Rigor

Available high-resolution structures of F-actin, myosin subfragment 1 (S1), and their complex, actin-S1, were used to calculate a 2D x-ray diffraction pattern from skeletal muscle in rigor. Actin sites occupied by myosin heads were chosen using a “principle of minimal elastic distortion energy” so that the 3D actin labeling pattern in the A-band of a sarcomere was determined by a single parameter. Computer calculations demonstrate that the total off-meridional intensity of a layer line does not depend on disorder of the filament lattice. The intensity of the first actin layer A1 line is independent of tilting of the “lever arm” region of the myosin heads. Myosin-based modulation of actin labeling pattern leads not only to the appearance of the myosin and “beating” actin-myosin layer lines in rigor diffraction patterns, but also to changes in the intensities of some actin layer lines compared to random labeling. Results of the modeling were compared to experimental data obtained from small bundles of rabbit muscle fibers. A good fit of the data was obtained without recourse to global parameter search. The approach developed here provides a background for quantitative interpretation of the x-ray diffraction data from contracting muscle and understanding structural changes underlying muscle contraction.

Kinetic Effects of Myosin Regulatory Light Chain Phosphorylation on Skeletal Muscle Contraction

Kinetic analysis of contracting fast and slow rabbit muscle fibers in the presence of the tension inhibitor 2,3-butanedione monoxime suggests that regulatory light chain (RLC) phosphorylation up-regulates the flux of weakly attached cross-bridges entering the contractile cycle by increasing the actin-catalyzed release of phosphate from myosin. This step appears to be separate from earlier Ca2+ regulated steps. Small step-stretches of single skinned fibers were used to study the effect of phosphorylation on fiber mechanics. Subdivision of the resultant tension transients into the Huxley-Simmons phases 1, 2fast, 2slow, 3, and 4 reveals that phosphorylation reduces the normalized amplitude of the delayed rise in tension (stretch activation response) by decreasing the amplitudes of phase 3 and, to a lesser extent, phase 2slow. In slow fibers, the RLC P1 isoform phosphorylates at least 4-fold faster than the P2 isoform, complicating the role of RLC phosphorylation in heart and slow muscle. We discuss the functional relevance of the regulation of stretch activation by RLC phosphorylation for cardiac and other oscillating muscles and speculate how the interaction of the two heads of myosin could account for the inverse effect of Ca2+ levels on isometric tension and rate of force redevelopment (kTR).

Backward Movements of Cross-Bridges by Application of Stretch and by Binding of MgADP to Skeletal Muscle Fibers in the Rigor State as Studied by X-Ray Diffraction

The effects of the applied stretch and MgADP binding on the structure of the actomyosin cross-bridges in rabbit and/or frog skeletal muscle fibers in the rigor state have been investigated with improved resolution by x-ray diffraction using synchrotron radiation. The results showed a remarkable structural similarity between cross-bridge states induced by stretch and MgADP binding. The intensities of the 14.4- and 7.2-nm meridional reflections increased by ∼23 and 47%, respectively, when 1 mM MgADP was added to the rigor rabbit muscle fibers in the presence of ATP-depletion backup system and an inhibitor for muscle adenylate kinase or by ∼33 and 17%, respectively, when rigor frog muscle was stretched by ∼4.5% of the initial muscle length. In addition, both MgADP binding and stretch induced a small but genuine intensity decrease in the region close to the meridian of the 5.9-nm layer line while retaining the intensity profile of its outer portion. No appreciable influence was observed in the intensities of the higher order meridional reflections of the 14.4-nm repeat and the other actin-based reflections as well as the equatorial reflections, indicating a lack of detachment of cross-bridges in both cases. The changes in the axial spacings of the actin-based and the 14.4-nm-based reflections were observed and associated with the tension change. These results indicate that stretch and ADP binding mediate similar structural changes, being in the correct direction to those expected for that the conformational changes are induced in the outer portion distant from the catalytic domain of attached cross-bridges. Modeling of conformational changes of the attached myosin head suggested a small but significant movement (about 10–20°) in the light chain-binding domain of the head toward the M-line of the sarcomere. Both chemical (ADP binding) and mechanical (stretch) intervensions can reverse the contractile cycle by causing a backward movement of this domain of attached myosin heads in the rigor state.

Examining a transparent object with a linnik tomographic microscope

Various applications are described for a Linnik tomographic microscope to biological and engineering phase objects. An interferometric microscope has been applied to human lymphocytes and to the measurement of birefringence on rabbit muscle fibers, as well as in nondestructive quality testing on microlens rasters and embossed holograms.

Force generation upon hydrostatic pressure release in tetanized intact frog muscle fibres.

Single intact muscle fibres isolated from the tibialis anterior muscle of the frog were exposed to hydrostatic pressures of 1-10 MPa, at 2-4 degrees C and sarcomere length of 2.1-2.2 microm. The pressure was rapidly released (ca. 1 ms) to atmospheric level (0.1 MPa) during the plateau of a tetanic contraction (Po) and the resultant tension (= force) transient examined. The pressure release induced tension transient consisted of an initial tension drop coincident with pressure release (ca. 4% Po per MPa, Phase 1), followed by a rapid recovery (Phase 2a) and a slower rise of tension (Phase 2b). Phase 1 was partly due to a length release at fibre ends (ca. 0.1 nm per half-sarcomere per MPa) induced by pressure-release effects on the steel chamber and fibre attachments, and partly due to 'expansion' upon pressure release within muscle fibre (ca. 0.2 nm per half-sarcomere per MPa), probably in the myofilaments and cross-bridges. The rate of tension recovery during phase 2a (ca. 600/s) was comparable to that of the quick tension recovery (T1-T2 transition) reported from moderately fast small length releases; the time course of Phase 2b (rate ca. 40/s) was similar to the late phase of tension rise in a tetanus, and hence compared with Phase 4 (T4) of a length release tension transient. Results are compared with the previously reported findings from analogous experiments on Ca2+ -activated skinned (rabbit) muscle fibres.

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.

ATPase kinetics on activation of rabbit and frog permeabilized isometric muscle fibres: a real time phosphate assay.

1. The rate of appearance of inorganic phosphate (Pi) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP) in the presence and absence of Ca2+. Pi appearance was monitored from the fluorescence signal of a Pi-sensitive probe, MDCC-PBP, a coumarin-labelled A197C mutant of the phosphate-binding protein from Escherichia coli. Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems. The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH-linked coupled enzyme assay. 2. On photolysis of NPE-caged ATP in the presence of Ca2+ at 20 degrees C, the fluorescence increase of MDCC-PBP was non-linear with time. ATPase activity was 41 s-1 in the first turnover based on a myosin subfragment 1 concentration of 150 microM. This was calculated from a linear regression of the fluorescence signal reporting 20-150 microM of Pi release. Tension was at 67% of its isometric level by the time 150 microM Pi was released. ATPase activities were 36 and 31 s-1 for Pi released in the ranges of 150-300 microM and 300-450 microM, respectively. The ATPase activity had a Q10 value of 2.9 based on measurements at 5, 12 and 20 degrees C. 3. An NADH-linked assay showed the ATPase activity had a lower limit of 12.7 s-1 at 20 degrees C. The response to photolytic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions. Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s-1. 4. On photolysis of NPE-caged ATP in the absence of Ca2+ the ATPase activity was 0.11 s-1 at 20 degrees C with no discernible rapid transient phase of Pi release during the first turnover of the ATPase. 5. To avoid the rigor state, the ATPase rate in the presence of Ca2+ was also measured on activation from the relaxed state by photolytic release of Ca2+ from a caged Ca2+ compound, nitrophenyl-EGTA. At 5 degrees C the ATPase rate was 5.8 and 4.0 s-1 in the first and second turnovers, respectively. These rates are comparable to those when NPE-caged ATP was used. 6. The influence of ADP and Pi on the ATPase activities was measured using the MDCC-PBP and NADH-linked assays, respectively. ADP (0.5 mM) decreased the initial ATPase rate by 23%. Pi (10 mM) had no significant effect. Inhibition by ADP, formed during ATP hydrolysis, contributed to the decrease of ATPase activity with time. 7. The MDCC-PBP assay and NPE-caged ATP were used to measure the ATPase rate in single permeabilized muscle fibres of the semitendinosus muscle of the frog. At 5 degrees C in the presence of Ca2+ the ATPase activity was biphasic being 15.0 s-1 during the first turnover (based on 180 microM myosin subfragment 1). Tension was 74% of its isometric level by the time 180 microM Pi was released. During the third turnover the ATPase rate decreased to about 20% of that during the first turnover. 8. ATPase activity in isometric rabbit muscle fibres during the first few turnovers is about an order of magnitude greater than that when a steady state is reached. Possible reasons and the consequences for understanding the mechanism of muscular contraction are discussed.

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.

Small-Angle X-ray Diffraction of Muscle Using Undulator Radiation from the Tristan Main Ring at KEK

Time-resolved X-ray diffraction of muscle has demanded ever-increasing flux into small sample volumes with low beam divergence. Results are reported of static and time-resolved small-angle X-ray diffraction studies on muscle fibers using a hard X-ray undulator installed in the Tristan main ring at KEK, Tsukuba, Japan, as an innovative source of synchrotron radiation more intense and better collimated than that available with the Photon Factory bending-magnet beamline. Static studies used the low divergence of the source to obtain detailed high-quality diffraction patterns of stable muscle states. The diffraction patterns from live skeletal muscles showed the numerous (over 100) meridional reflections. The well collimated beam from the undulator made it possible to clearly resolve, with an angular resolution of ca 700 nm, the closely spaced diffraction peaks arising from the two halves of the thick filaments centred on the M lines in a sarcomere, in addition, the diffraction peaks from the thin filaments on opposite sides of the Z bands could be resolved with an angular resolution of ca 1000 nm. The detailed structure of the meridional pattern defines the nature of the molecular packing in the thick and thin filaments. Time-resolved experiments using a focusing mirror aimed to prove cross-bridge states in striated muscle fibers by collecting X-ray diffraction data at a 0.185 ms time resolution from sinusoidally oscillating chemically skinned rabbit muscle fibers during active contraction and in rigor. When sinusoidal length changes at 500 Hz with a peak-to-peak amplitude of 0.6% of the muscle length were applied to a small fiber bundle, the tension showed a simple elastic response during the length oscillation. In the active muscle the intensity of the 14.5 nm myosin-based meridional reflection changed out of phase with the tension change during the oscillating length change. In contrast, in the rigor muscle it occurred in phase with the tension change. The high time-resolved experiments provide an insight into the coupling between conformational changes and force generation of the actomyosin cross-bridges. These studies provide a preview of the expected gains for muscle studies from the more widespread use of undulator radiation at third-generation synchrotron sources.

Endothermic force generation in fast and slow mammalian (rabbit) muscle fibers

Isometric tension responses to rapid temperature jumps (T-jumps) of 3-7 degrees C were examined in single skinned fibers isolated from rabbit psoas (fast) and soleus (slow) muscles. T-jumps were induced by an infrared laser pulse (wavelength 1.32 microns, pulse duration 0.2 ms) obtained from a Nd-YAG laser, which heated the fiber and bathing buffer solution in a 50-microliter trough. After a T-jump, the temperature near the fiber remained constant for approximately 0.5 s, and the temperature could be clamped for longer periods by means of Peltier units assembled on the back trough wall. A T-jump produced a step decrease in tension in both fast and slow muscle fibers in rigor, indicating thermal expansion. In maximally Ca-activated (pCa approximately 4) fibers, the increase of steady tension with heating (3-35 degrees C) was approximately sigmoidal, and a T-jump at any temperature induced a more complex tension transient than in rigor fibers. An initial (small amplitude) step decrease in tension followed by a rapid recovery (tau(1); see Davis and Harrington, 1993) was seen in some records from both fiber types, which presumably was an indirect consequence of thermal expansion. The net rise in tension after a T-jump was biexponential, and its time course was characteristically different in the two fibers. At approximately 12 degrees C the reciprocal time constants for the two exponential components (tau(2) and tau(3), respectively, were approximately 70.s(-1) and approximately 15.s(-1) in fast fibers and approximately 20.s(-1) and approximately 3.s(-1) in slow fibers. In both fibers, tau(2) ("endothermic force regeneration") became faster with an increase in temperature. Furthermore, tau(3) was temperature sensitive in slow fibers but not in fast fibers. The results are compared and contrasted with previous findings from T-jump experiments on fast fibers. It is observed that the fast/slow fiber difference in the rate of endothermic force generation (three- to fourfold) is considerably smaller than the reported differences in the "phosphate release steps" (> 30-fold).

Influence of physiological L(+)-lactate concentrations on contractility of skinned striated muscle fibers of rabbit.

These experiments investigated the effects of physiological concentrations of L(+)-lactate on the contractility of chemically skinned rabbit fast-twitch psoas, slow-twitch soleus, and cardiac muscles at pH 7.L(+)-Lactate depressed maximal calcium-activated force (Fmax) of all muscles studied within the range of 5-20 (slow-twitch muscle) or 5-25 mM (fast-twitch and cardiac muscles). Fmax of fast-twitch fibers was inhibited to the greatest degree (9% in K2 creatine phosphate solutions). In all of these muscle types, Fmax returned to control levels as L(+)-lactate was increased to 30-50 mM. Substitution of neither D-lactate nor propionate for L(+)-lactate significantly altered Fmax. In addition, with the exception of fast-twitch muscle (where the Hill coefficient decreased), L(+)-lactate concentrations, which maximally inhibited Fmax, did not affect the force vs. pCa relationship of muscles tested. These results demonstrate that L(+)-lactate significantly contributes to the depression of muscle function noted during lactic acidosis, directly inhibiting Fmax of the contractile apparatus. This contribution is maximal in fast-twitch muscle where L(+)-lactate is responsible for as much as one-third of the depressant effect on Fmax of the contractile apparatus noted during lactic acidosis.

Regulation of the cross-bridge transition from a weakly to strongly bound state in skinned rabbit muscle fibers

The regulation of cross-bridge transition from weakly attached to force-bearing states was studied at 10 degrees C in skinned muscle fibers by measuring the rate of force development after a quick release-restretch cycle (ktr), the rate of force decline (kPi) after photogeneration of Pi from caged Pi, and stiffness in the presence and absence of an inhibitor of strong cross-bridge formation, 2,3-butanedione monoxime (BDM). Both BDM and Pi suppressed force more than stiffness. However, reduction of Ca2+ suppressed force and stiffness in a parallel fashion. Both ktr and kPi were reversibly reduced (by 30-35%) in 3 mM BDM, but both were increased by increasing Pi concentration. Reduction of Ca2+ concentration to match the force seen in 3 mM BDM had no effect on kPi but decreased ktr by 85%. These results are inconsistent with cross-bridge models undergoing the transition from a weakly bound to a force-generating state in a single step but are consistent with a model having two steps, one of which is controlled by pCa.

Force generation and work production by covalently cross-linked actin-myosin cross-bridges in rabbit muscle fibers

To separate a fraction of the myosin cross-bridges that are attached to the thin filaments and that participate in the mechanical responses, muscle fibers were cross-linked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and then immersed in high-salt relaxing solution (HSRS) of 0.6 M ionic strength for detaching the unlinked myosin heads. The mechanical properties and force-generating ability of the cross-linked cross-bridges were tested with step length changes (L-steps) and temperature jumps (T-jumps) from 6-10 degrees C to 30-40 degrees C. After partial cross-linking, when instantaneous stiffness in HSRS was 25-40% of that in rigor, the mechanical behavior of the fibers was similar to that during active contraction. The kinetics of the T-jump-induced tension transients as well as the rate of the fast phase of tension recovery after length steps were close to those in unlinked fibers during activation. Under feedback force control, the T-jump initiated fiber shortening by up to 4 nm/half-sarcomere. Work produced by a cross-linked myosin head after the T-jump was up to 30 x 10(-21) J. When the extent of cross-linking was increased and fiber stiffness in HSRS approached that in rigor, the fibers lost their viscoelastic properties and ability to generate force with a rise in temperature.