Albert M. Gordon

Calcium regulation of skeletal muscle thin filament motility in vitro

Using an in vitro motility assay, we have investigated Ca2+ regulation of individual, regulated thin filaments reconstituted from rabbit fast skeletal actin, troponin, and tropomyosin. Rhodamine-phalloidin labeling was used to visualize the filaments by epifluorescence, and assays were conducted at 30 degrees C and at ionic strengths near the physiological range. Regulated thin filaments exhibited well-regulated behavior when tropomyosin and troponin were added to the motility solutions because there was no directed motion in the absence of Ca2+. Unlike F-actin, the speed increased in a graded manner with increasing [Ca2+], whereas the number of regulated thin filaments moving was more steeply regulated. With increased ionic strength, Ca2+ sensitivity of both the number of filaments moving and their speed was shifted toward higher [Ca2+] and was steepest at the highest ionic strength studied (0.14 M gamma/2). Methylcellulose concentration (0.4% versus 0.7%) had no effect on the Ca2+ dependence of speed or number of filaments moving. These conclusions hold for five different methods used to analyze the data, indicating that the conclusions are robust. The force-pCa relationship (pCa = -log10[Ca2+]) for rabbit psoas skinned fibers taken under similar conditions of temperature and solution composition (0.14 M gamma/2) paralleled the speed-pCa relationship for the regulated filaments in the in vitro motility assay. Comparison of motility results with the force-pCa relationship in fibers suggests that relatively few cross-bridges are needed to make filaments move, but many have to be cycling to make the regulated filament move at maximum speed.

Thin filament near-neighbour regulatory unit interactions affect rabbit skeletal muscle steady-state force-Ca2+ relations

The role of cooperative interactions between individual structural regulatory units (SUs) of thin filaments (7 actin monomers : 1 tropomyosin : 1 troponin complex) on steady‐state Ca2+‐activated force was studied. Native troponin C (TnC) was extracted from single, de‐membranated rabbit psoas fibres and replaced by mixtures of purified rabbit skeletal TnC (sTnC) and recombinant rabbit sTnC (D27A, D63A), which contains mutations that disrupt Ca2+ coordination at N‐terminal sites I and II (xxsTnC). Control experiments in fibres indicated that, in the absence of Ca2+, both sTnC and xxsTnC bind with similar apparent affinity to sTnC‐extracted thin filaments. Endogenous sTnC‐extracted fibres reconstituted with 100 % xxsTnC did not develop Ca2+‐activated force. In fibres reconstituted with mixtures of sTnC and xxsTnC, maximal Ca2+‐activated force increased in a greater than linear manner with the fraction of sTnC. This suggests that Ca2+ binding to functional Tn can spread activation beyond the seven actins of an SU into neighbouring units, and the data suggest that this functional unit (FU) size is up to 10–12 actins. As the number of FUs was decreased, Ca2+ sensitivity of force (pCa50) decreased proportionally. The slope of the force‐pCa relation (the Hill coefficient, nH) also decreased when the reconstitution mixture contained < 50 % sTnC. With 15 % sTnC in the reconstitution mixture, nH was reduced to 1.7 ± 0.2, compared with 3.8 ± 0.1 in fibres reconstituted with 100 % sTnC, indicating that most of the cooperative thin filament activation was eliminated. The results suggest that cooperative activation of skeletal muscle fibres occurs primarily through spread of activation to near‐neighbour FUs along the thin filament (via head‐to‐tail tropomyosin interactions).

Kinetic Studies of Calcium Binding to the Regulatory Site of Troponin C from Cardiac Muscle

We have studied the kinetics of the structural transitions induced by calcium binding to the single, regulatory site of cardiac troponin C by measuring the rates of calcium-mediated fluorescence changes with a monocysteine mutant of the protein (C35S) specifically labeled at Cys-84 with the fluorescent probe 2-[4′-(iodoacetamido)anilino]naphthalene-6-sulfonic acid. At 4°C, the binding kinetics determined in the presence of Mg2+ was resolved into two phases with positive amplitude, which were completed in less than 100 ms. The rate of the fast phase increased linearly with [Ca2+] reaching a maximum of ∼590 s−1, and that of the slow phase was approximately 100 s−1 and did not depend on Ca2+ concentration. Dissociation of bound Ca2+ from the regulatory site occurred with a rate of 102 s−1, whereas the dissociation from the two high affinity sites was about two orders of magnitude slower. These results are consistent with the following scheme for the binding of Ca2+ to the regulatory site: where the asterisks denote states with enhanced fluorescence. The apparent second-order rate constant for calcium binding is Kok1 = 1.4 × 108M−1 s−1. The two first-order transitions occur with observed rates of k1 + k−1 ≈ 590 s−1 and k2 + k−2 ≈ 100 s−1, and the binding of Ca2+ to the regulatory site is not a simple diffusion-controlled reaction. These transitions provide the first information on the rates of Ca2+-induced conformational changes involving helix movements in the regulatory domain.

Length and myofilament spacing-dependent changes in calcium sensitivity of skeletal fibres: effects of pH and ionic strength

SummaryThe calcium sensitivity of force was measured in glycerinated rabbit psoas fibres at sarcomere lengths (SL) from 2.3 to 3.4 Μm. Increasd SL caused calcium sensitivity to increase and the slope of force-calcium relations to decrease. We have hypothesized that length-dependent changes in myofilament lattice spacing and the presence of fixed charge on the myofilaments are important in determining calcium sensitivity. Lattice spacing changes were monitored by measuring fibre diameter (D). D was decreased by increasing SL, decreasing bathing solution pH and by osmotic compression with 3% PVP. 3% PVP caused D to decrease by about 15% at all SLs and pH values tested. Force-calcium relations were measured at different SLs and pH values, with and without 3% PVP in the bathing solutions. At all pH values D at SL 2.3 Μm with 3% PVP was comparable to the value at 3.4 Μm, without PVP. At pH 7.5 and 7.0 calcium sensitivity was about the same at both SL, although the slope of the force-calcium relation was less at longer SL. The similarity of the calcium sensitivity at the same D, but much different SL, indicates that lattice spacing is important in determining calcium sensitivity, while SL and the degree of myofilament overlap are important in determing the slope of force-calcium relations. In order to test for the role of myofilament charge in determining calcium sensitivity, pH and ionic strength were varied. Decreasing pH caused decreased maximum force and calcium sensitivity. In addition, the influence of SL on calcium sensitivity decreased as pH was lowered, with minimal SL dependence at pH 5.5; even though lattice spacing still decreased with increasing SL. When D was decreased with PVP, calcium sensitivity increased at all SLs in pH 7.5 and 7.0 while the same lattice spacing changes at pH 6.0 and 5.5 resulted in greatly reduced shifts in calcium sensitivity. These results indicate that the effect of lattice spacing on calcium sensitivity depends on myofilament charge. At pH 6.0, even though osmotic compression of the lattice has no effect, increasing SL causes about half the shift in calcium sensitivity seen at pH 7.0. Lowering ionic strength from 200 to 110 mM caused an increase in both the magnitude and length dependence of calcium sensitivity at pH 7.0, while at pH 5.5 both decreased. Although changes in pH and ionic strength can alter crossbridge binding and kinetics, and the binding of calcium to troponin, as well as the magnitude of myofilament charge, the results indicate the importance of both lattice spacing and fixed myofilament charge in determining contractile sensitivity to calcium.

Effects of inorganic phosphate analogues on stiffness and unloaded shortening of skinned muscle fibres from rabbit.

1. We examined the effects of aluminofluoride (AlFx) and orthovanadate (Vi), tightly binding analogues of orthophosphate (Pi), on the mechanical properties of glycerinated fibres from rabbit psoas muscle. Maximum Ca(2+)‐activated force, stiffness, and unloaded shortening velocity (Vus) were measured under conditions of steady‐state inhibition (up to 1 mM of inhibitor) and during the recovery from inhibition. 2. Stiffness was measured using either step or sinusoidal (1 kHz) changes in fibre length. Sarcomere length was monitored continuously by helium‐neon laser diffraction during maximum Ca2+ activation. Stiffness was determined from the changes in sarcomere length and the corresponding changes in force. Vus was measured using the slack test method. 3. AlF chi and Vi each reversibly inhibited force, stiffness and Vus. Actively cycling cross‐bridges were required for reversal of these inhibitory effects. Recovery from inhibition by AlF chi was 3‐ to 4‐fold slower than that following removal of V1. 4. At various degrees of inhibition, AlF chi and Vi both inhibited steady‐state isometric force more than either Vus or stiffness. For both AlF chi and Vi, the relatively greater inhibition of force over stiffness persisted during recovery from steady‐state inhibition. We interpret these results to indicate that the cross‐bridges with AlF chi or Vi bound are analogous to those which occur early in the cross‐bridge cycle.

A simple model with myofilament compliance predicts activation-dependent crossbridge kinetics in skinned skeletal fibers.

The contribution of thick and thin filaments to skeletal muscle fiber compliance has been shown to be significant. If similar to the compliance of cycling cross-bridges, myofilament compliance could explain the difference in time course of stiffness and force during the rise of tension in a tetanus as well as the difference in Ca(2+) sensitivity of force and stiffness and more rapid phase 2 tension recovery (r) at low Ca(2+) activation. To characterize the contribution of myofilament compliance to sarcomere compliance and isometric force kinetics, the Ca(2+)-activation dependence of sarcomere compliance in single glycerinated rabbit psoas fibers, in the presence of ATP (5.0 mM), was measured using rapid length steps. At steady sarcomere length, the dependence of sarcomere compliance on the level of Ca(2+)-activated force was similar in form to that observed for fibers in rigor where force was varied by changing length. Additionally, the ratio of stiffness/force was elevated at lower force (low [Ca(2+)]) and r was faster, compared with maximum activation. A simple series mechanical model of myofilament and cross-bridge compliance in which only strong cross-bridge binding was activation dependent was used to describe the data. The model fit the data and predicted that the observed activation dependence of r can be explained if myofilament compliance contributes 60-70% of the total fiber compliance, with no requirement that actomyosin kinetics be [Ca(2+)] dependent or that cooperative interactions contribute to strong cross-bridge binding.

UNLOADED SHORTENING OF SKINNED MUSCLE FIBERS FROM RABBIT ACTIVATED WITH AND WITHOUT CA2

Unloaded shortening velocity (VUS) was determined by the slack method and measured at both maximal and submaximal levels of activation in glycerinated fibers from rabbit psoas muscle. Graded activation was achieved by two methods. First, [Ca2+] was varied in fibers with endogenous skeletal troponin C (sTnC) and after replacement of endogenous TnC with either purified cardiac troponin C (cTnC) or sTnC. Alternatively, fibers were either partially or fully reconstituted with a modified form of cTnC (aTnC) that enables force generation and shortening in the absence of Ca2+. Uniformity of the distribution of reconstituted TnC across the fiber radius was evaluated using fluorescently labeled sTnC and laser scanning fluorescence confocal microscopy. Fiber shortening was nonlinear under all conditions tested and was characterized by an early rapid phase (VE) followed by a slower late phase (VL). In fibers with endogenous sTnC, both VE and VL varied with [Ca2+], but VE was less affected than VL. Similar results were obtained after extraction of TnC and reconstitution with either sTnC or cTnC, except for a small increase in the apparent activation dependence of VE. Partial activation with aTnC was obtained by fully extracting endogenous sTnC followed by reconstitution with a mixture of aTnC and cTnC (aTnC:cTnC molar ratio 1:8.5). At pCa 9.2, VE and VL were similar to those obtained in fibers reconstituted with sTnC or cTnC at equivalent force levels. In these fibers, which contained aTnC and cTnC, VE and VL increased with isometric force when [Ca2+] was increased from pCa 9.2 to 4.0. Fibers that contained a mixture of a TnC and cTnC were then extracted a second time to selectively remove cTnC. In fibers containing aTnC only, VE and VL were proportional to the resulting submaximal isometric force compared with maximum Ca(2+)-activated control. With aTnC alone, force, VE, and VL were not affected by changes in [Ca2+]. The similarity of activation dependence of VUS whether fibers were activated in a Ca(2+)-sensitive or -insensitive manners implies that VUS is determined by the average level of thin filament activation and that, with sTnC or cTnC, VUS is affected by Ca2+ binding to TnC only.

Models of calcium activation account for differences between skeletal and cardiac force redevelopment kinetics

To explain observed differences in the activation dependence of force redevelopment kinetics between cardiac and skeletal muscle, two numerical models of contractile regulation by Ca2+ were investigated. Ca2+ binding and force production were each modelled as two-state processes with forward and reverse rate constants taken from the literature. The first model incorporates four possible thin-filament states. In the second model Ca2+ is assumed not to dissociate from a thin-filament unit in the force-generating state, resulting in three states. The four-state model can account for the activation dependence of the rate constant of tension redevelopment (ktr) seen in skeletal muscle, without requiring that Ca2+ directly modulates the kinetics of any step in the cross-bridge cycle. Using identical kinetic parameters, the three-state model shows no activation dependence of ktr, consistent with our results in cardiac muscle. Following a step increase in [Ca2+], the rate of rise in tension (as described by the rate constant kCa) varies with the final [Ca2+] for both models, consistent with experimental results from skeletal and cardiac muscle. These numerical models demonstrate that experimental measurements thought to reveal changes in kinetic parameters may simply reflect coupling between the two kinetic processes of Ca2+ binding and force generation. Furthermore, the models present possible differences in the Ca2+ activation scheme between cardiac and skeletal muscle which can account for the contrasting activation dependencies of force redevelopment kinetics

Skeletal muscle regulatory proteins enhance F-actin in vitro motility.

Using an in vitro motility assay, we have investigated the effects of rabbit skeletal muscle regulatory proteins, troponin and tropomyosin, on the gliding of F-actin filaments or F-actin filaments containing these regulatory proteins. We demonstrate that Ca2+ does not affect the motility of F-actin gliding on HMM, but does in the presence of skeletal muscle tropomyosin and troponin. We conclude that Ca2+ affects motility through troponin because, like F-actin, F-actin-Tm filaments show no Ca(2+)-dependence to their gliding speeds. Furthermore, there is a large enhancement of the gliding speed (about 75%) in the presence of skeletal muscle tropomyosin, troponin + saturating Ca2+ over that seen with F-actin filaments. This enhancement is not due to the action of tropomyosin alone as skeletal muscle tropomyosin without troponin enhances the speed little (about 5%) over that of F-actin. Thus troponin confers Ca2+ sensitivity to the motility and, additionally, potentiates motility greatly along with tropomyosin in the presence of saturating Ca2+. When [HMM] is varied, the decline in speed of F-actin seen at low HMM density is changed little by tropomyosin in the F-actin-Tm filaments. These data show that the skeletal regulatory proteins interact with F-actin to enhance the interaction with HMM particularly in the presence of troponin and saturating Ca2+ and enhance the gliding speed in the in vitro motility assay as they potentiate the ATPase activity in the isolated proteins. This enhancement of speed in the motility assay cannot be ascribed to tropomyosin alone.

Influence of Ca2+ on force redevelopment kinetics in skinned rat myocardium

The influence of Ca2+ on isometric force kinetics was studied in skinned rat ventricular trabeculae by measuring the kinetics of force redevelopment after a transient decrease in force. Two protocols were employed to rapidly detach cycling myosin cross-bridges: a large-amplitude muscle length ramp followed by a restretch back to the original length or a 4% segment length step. During the recovery of force, the length of the central region of the muscle was controlled by using a segment marker technique and software feedback control. Tension redevelopment was fit by a rising exponential governed by the rate constant ktr for the ramp/restretch protocol and kstep for the step protocol. ktr and kstep averaged 7.06 s-1 and 15.7 s-1, respectively, at 15 degrees C; neither ktr nor kstep increased with the level of Ca2+ activation. Similar results were found at submaximum Ca2+ levels when sarcomere length control by laser diffraction was used. The lack of activation dependence of ktr contrasts with results from fast skeletal fibers, in which ktr varies 10-fold from low to high activation levels, and suggests that Ca2+ does not modulate the kinetics of cross-bridge attachment or detachment in mammalian cardiac muscle.