Rabbit Psoas Fibers Research Articles

Passive force enhancement is not abolished by shortening of single rabbit psoas fibres

Passive force enhancement is defined as the increase in steady-state passive force following deactivation of an actively stretched muscle compared to the corresponding passive force following passive stretching of the muscle. Passive force enhancement has been associated with contributing to the residual force enhancement property, providing stability to sarcomeres, and preventing sarcomeres from over-stretching during eccentric muscle action. Despite its functional importance, the molecular mechanisms underlying passive force enhancement remain unknown. Specifically, it remains unknown how passive force enhancement develops and how it is abolished. Incidental observations on cat soleus muscles led to the speculation that passive force enhancement is abolished when the actively stretched muscle is deactivated and then passively shortened to its pre-stretched length. Here, we tested this hypothesis using skinned fibres from rabbit psoas and rejected it. Rather, we found that passive force enhancement increased following shortening of the fibres to their pre-stretched length (2.4 µm), and furthermore, that the passive force enhancement increased by 70–106% when the shortening and subsequent stretch to the original length (3.6 µm) increased in duration (200 ms, 6 s, and 14 s). These results indicate that passive force enhancement increases during a shortening–stretch cycle, and that this increase is time-dependent. We propose that this increase in passive force enhancement is caused by titin; specifically, with a refolding of titin’s immunoglobulin domains that were unfolded during the active fibre stretching that produced the residual and passive force enhancement. Molecular level experiments are required to test this proposal.

Energy Cost of Force Production After a Stretch-Shortening Cycle in Skinned Muscle Fibers: Does Muscle Efficiency Increase?

Muscle force is enhanced during shortening when shortening is preceded by an active stretch. This phenomenon is known as the stretch-shortening cycle (SSC) effect. For some stretch-shortening conditions this increase in force during shortening is maintained following SSCs when compared to the force following a pure shortening contraction. It has been suggested that the residual force enhancement property of muscles, which comes into play during the stretch phase of SSCs may contribute to the force increase after SSCs. Knowing that residual force enhancement is associated with a substantial reduction in metabolic energy per unit of force, it seems reasonable to assume that the metabolic energy cost per unit of force is also reduced following a SSC. The purpose of this study was to determine the energy cost per unit of force at steady-state following SSCs and compare it to the corresponding energy cost following pure shortening contractions of identical speed and magnitude. We hypothesized that the energy cost per unit of muscle force is reduced following SSCs compared to the pure shortening contractions. For the SSC tests, rabbit psoas fibers (n = 12) were set at an average sarcomere length (SL) of 2.4 μm, activated, actively stretched to a SL of 3.2 μm, and shortened to a SL of 2.6 or 3.0 μm. For the pure shortening contractions, the same fibers were activated at a SL of 3.2 μm and actively shortened to a SL of 2.6 or 3.0 μm. The amount of ATP consumed was measured over a 40 s steady-state total isometric force following either the SSCs or the pure active shortening contractions. Fiber stiffness was determined in an additional set of 12 fibers, at steady-state for both experimental conditions. Total force, ATP consumption, and stiffness were greater following SSCs compared to the pure shortening contractions, but ATP consumption per unit of force was the same between conditions. These results suggest that the increase in total force observed following SSCs was achieved with an increase in the proportion of attached cross-bridges and titin stiffness. We conclude that muscle efficiency is not enhanced at steady-state following SSCs.

Effects of myosin inhibitors on the X-ray diffraction patterns of relaxed and calcium-activated rabbit skeletal muscle fibers.

We studied the effect of myosin inhibitors, N-benzyl-p-toluenesulfonamide (BTS), blebbistatin, and butanedione monoxime (BDM) on X-ray diffraction patterns from rabbit psoas fibers under relaxing and contracting conditions. The first two inhibitors suppressed the contractile force almost completely at a 100 μM concentration, and a similar effect was obtained at 50 mM for BDM. However, still substantial changes were observed in the diffraction patterns upon calcium-activation of inhibited muscle fibers. (1) The 2nd actin layer-line reflection was enhanced normally, indicating that calcium binding to troponin and the subsequent movement of tropomyosin are not inhibited, (2) the myosin layer-line reflections became much weaker, and (3) the 1,1/1,0 intensity ratio of the equatorial reflections was increased. The observations (2) and (3) indicate that, even in the presence of the inhibitors at a saturating concentration, myosin heads leave the helix on the thick filaments and approach the thin filaments. Interestingly, the d1,0 spacing of the filament lattice remained unchanged upon activation of inhibited fibers, in contrast to the case of normal activation in which the spacing is decreased. This suggests that the normal activated myosin heads exert a pull in both axial and radial directions, but in the presence of the inhibitors, the pull is suppressed, and as a result, the heads simply bind to actin without exerting any force. The results support the idea that the inhibitors do not block the myosin binding to actin, but block the step of force-producing transition of the bound actomyosin complex.

High ionic strength depresses muscle contractility by decreasing both force per cross-bridge and the number of strongly attached cross-bridges.

An increase in ionic strength (IS) lowers Ca(2+) activated tension in muscle fibres, however, its molecular mechanism is not well understood. In this study, we used single rabbit psoas fibres to perform sinusoidal analyses. During Ca(2+) activation, the effects of ligands (ATP, Pi, and ADP) at IS ranging 150-300 mM were studied on three rate constants to characterize elementary steps of the cross-bridge cycle. The IS effects were studied because a change in IS modifies the inter- and intra-molecular interactions, hence they may shed light on the molecular mechanisms of force generation. Both the ATP binding affinity (K1) and the ADP binding affinity (K 0) increased to 2-3x, and the Pi binding affinity (K5) decreased to 1/2, when IS was raised from 150 to 300 mM. The effect on ATP/ADP can be explained by stereospecific and hydrophobic interaction, and the effect on Pi can be explained by the electrostatic interaction with myosin. The increase in IS increased cross-bridge detachment steps (k2 and k-4), indicating that electrostatic repulsion promotes these steps. However, IS did not affect attachment steps (k-2 and k4). Consequently, the equilibrium constant of the detachment step (K2) increased by ~100%, and the force generation step (K4) decreased by ~30%. These effects together diminished the number of force-generating cross-bridges by 11%. Force/cross-bridge (T56) decreased by 26%, which correlates well with a decrease in the Debye length that limits the ionic atmosphere where ionic interactions take place. We conclude that the major effect of IS is a decrease in force/cross-bridge, but a decrease in the number of force generating cross-bridge also takes place. The stiffness during rigor induction did not change with IS, demonstrating that in-series compliance is not much affected by IS.

Calcium Sensitivity after Active Shortening in Rabbit Psoas Fibres

Introduction It has been shown that isometric force after active shortening of muscles is lower than the purely isometric force performed at the corresponding lengths. This property is termed force depression. Force depression has been observed in isolated muscle preparations and muscles in situ and in vivo (Rassier et al., J Applied Physiol 2004). The origin of force depression is still unknown. It has been suggested that active shortening may affect calcium sensitivity by changing calcium-troponin binding equilibrium leading to decreased force after active shortening (Ekelund and Edman, Acta Physiol Scand 1982). Our aim in the present study was to test whether calcium sensitivity was decreased after active shortening. Methods Experiments were performed using skinned fibres (n=7) isolated from rabbit psoas muscle. Calcium sensitivity was characterized by establishing the force-pCa curves for isometric reference contractions performed at an average sarcomere length of 2.4 µm and active shortening contractions from an average sarcomere length of 3.0 µm to an average sarcomere length of 2.4 µm. pCa50 and the coefficient of cooperativity (nH) were compared between reference and active shortening contractions. Results and Discussion pCa50 for the reference contractions was 5.99±0.02. No change in pCa50 was observed in the force depressed state (6.00±0.01). Furthermore, the coefficient of cooperativity did not change after active shortening (3.9±0.3 versus 4.3±0.3 for the reference and force depressed states respectively). These results suggest that calcium sensitivity does not decrease after active shortening and that the binding interactions between troponin and calcium are not altered in the force depressed state.

The Effects of Titin Degradation on Passive Stiffness Properties of Skinned Rabbit Psoas Fibers during Osmotic Compression

Muscle is disproportionately stiff over short stretch distances. This effect is largely calcium-sensitive, and can be eliminated in actively contracting skinned fibers by inhibiting crossbridge formation with pharmacological agents such as 2,3-butanedione monoxime (BDM). However, elevated short range stiffness can be induced in the absence of calcium in relaxed skinned muscle fibers by osmotic compression, an effect which is not attenuated by BDM. This study aimed to determine if titin, the primary source of tension and stiffness in relaxed muscle, could account for the elevated short range stiffness in osmotically compressed, relaxed skinned fibers. Accordingly, skinned rabbit psoas fibers (n=9) were held at 2.8 μm sarcomere length and osmotically compressed using 7.5% dextran-T-500 in calcium-free relaxing solution. Muscles were stretched by 2% (∼0.057 μm/sarcomere) over 0.66 s, and returned to initial length over another 0.66 s. Dextran treatment resulted in a 5.6 ± 0.6 fold increase in short range stiffness, with an apparent length limit of 4.5±0.2 nm per sarcomere (Phase 1). Beyond this limit (Phase 2), stiffness was not different (100.4±3.0%) than dextran-free stiffness. To selectively degrade titin, fibers were held in relaxing solution containing both dextran and trypsin (0.25 μg/mL) until mechanical failure, with periodic application of the stretch protocol. In the stretch immediately preceding failure, Phase 2 stiffness was 86±3% lower than initial values, while Phase 1 stiffness only decreased by 31±3%. Additionally, tension at peak stretch increased 12.5±2.1 μN (33±3%) upon osmotic compression, matching the 12.6±1.1 μN intercept of the Phase 2 regression line on the Δtension versus Δlength graph. Thus, short range stiffness appears additive to Phase 2, titin-based stiffness, and is best accounted for as a separate entity acting in parallel to titin.

A new mechanokinetic model for muscle contraction, where force and movement are triggered by phosphate release.

The atomic structure of myosin-S1 suggests that its working stroke, which generates tension and shortening in muscle, is triggered by the release of inorganic phosphate from the active site. This mechanism is the basis of a new mechanokinetic model for contractility, using the biochemical actomyosin ATPase cycle, strain-dependent kinetics and dimeric myosins on buckling rods. In this model, phosphate-dependent aspects of contractility arise from a rapid reversible release of phosphate from the initial bound state (A.M.ADP.Pi), which triggers the stroke. Added phosphate drives bound myosin towards this initial state, and the transient tension response to a phosphate jump reflects the rate at which it detaches from actin. Predictions for the tensile and energetic properties of striated muscle as a function of phosphate level, including the tension responses to length steps and Pi-jumps, are compared with experimental data from rabbit psoas fibres at 10 °C. The phosphate sensitivity of isometric tension is maximal when the actin affinity of M.ADP.Pi is near unity. Hence variations in actin affinity modulate the phosphate dependence of isometric tension, and may explain why phosphate sensitivity is temperature-dependent or absent in different muscles.

A New Theory on k(TR) with Series Elastic Elements

It has been known that when the length of active muscle fiber is released suddenly by ∼15% and re-stretched, tension falls to 0, then it redevelops exponentially with the rate constant ktr. The rise of tension has been interpreted to represent the force generation step (partial cross-bridge cycle). Here we propose a model, in which cross-bridges cycle many times in this period by stretching series elastic elements. One assumption needed is either the step size (η) or the stepping rate (v) decreases linearly with tension(F): η=η0{1-F(t)/F0} (1), or ν = ν0{1-F(t)/F0} (2), where F0 is isometric tension; η0 is step size and ν0 is the number of steps/sec without load. Eqs. 1 and 2 are mathematical representations of the Fenn effect. Distance travelled by cross-bridges in time dt is ηvdt, which stretches series elastic with stiffness σ. Thus, the increase in tension is: dF=σvηdt. By using Eq. 1 or 2, we get: dF/dt+kF=kF0 (3), where k=ση0ν0/F0 (4). By solving Eq. 3 with F(0)=0, we get F(t)=F0{1-exp(-kt)} (5). Eq. 5 is a good approximation of tension time course which deduced kTR. From Eq. 4, it can be concluded that kTR is proportionately related to the turn over rate (ν0). Because ATPase=M0ν0 (M0 is myosin concentration), k=ATPase•ση0/F0M0 (6). Our available data on rabbit psoas fibers on kTR and ATPase at 10°C–25°C demonstrate that these are proportionately related, as predicted by Eq. 6. Our data further demonstrate that kTR and 2πa (slowest rate constant of tension transients) are linearly related by 2πa=0.71kTR+0.89s−1, demonstrating that 2πa is also limited by the turn over rate. In the two state model with attachment rate (f) and detachment rate (g), the turnover rate is v0=fg/(f+g), hence kTR=(ση0/F0)(fg)/(f+g) (limited by a slow reaction). Therefore, kTR≠ f+g (limited by a fast reaction).

Filament Compliance Influences Cooperative Activation of Thin Filaments and the Dynamics of Force Production in Skeletal Muscle

Striated muscle contraction is a highly cooperative process initiated by Ca2+ binding to the troponin complex, which leads to tropomyosin movement and myosin cross-bridge (XB) formation along thin filaments. Experimental and computational studies suggest skeletal muscle fiber activation is greatly augmented by cooperative interactions between neighboring thin filament regulatory units (RU-RU cooperativity; 1 RU = 7 actin monomers+1 troponin complex+1 tropomyosin molecule). XB binding can also amplify thin filament activation through interactions with RUs (XB-RU cooperativity). Because these interactions occur with a temporal order, they can be considered kinetic forms of cooperativity. Our previous spatially-explicit models illustrated that mechanical forms of cooperativity also exist, arising from XB-induced XB binding (XB-XB cooperativity). These mechanical and kinetic forms of cooperativity are likely coordinated during muscle contraction, but the relative contribution from each of these mechanisms is difficult to separate experimentally. To investigate these contributions we built a multi-filament model of the half sarcomere, allowing RU activation kinetics to vary with the state of neighboring RUs or XBs. Simulations suggest Ca2+ binding to troponin activates a thin filament distance spanning 9 to 11 actins and coupled RU-RU interactions dominate the cooperative force response in skeletal muscle, consistent with measurements from rabbit psoas fibers. XB binding was critical for stabilizing thin filament activation, particularly at submaximal Ca2+ levels, even though XB-RU cooperativity amplified force less than RU-RU cooperativity. Similar to previous studies, XB-XB cooperativity scaled inversely with lattice stiffness, leading to slower rates of force development as stiffness decreased. Including RU-RU and XB-RU cooperativity in this model resulted in the novel prediction that the force-[Ca2+] relationship can vary due to filament and XB compliance. Simulations also suggest kinetic forms of cooperativity occur rapidly and dominate early to get activation, while mechanical forms of cooperativity act more slowly, augmenting XB binding as force continues to develop.

Cross-Bridge Populations and the Biphasic Time Course of Muscle Relaxation

A biphasic time course of relaxation follows Ca2+ removal from a contracting muscle fiber--a small linear decline is followed by a faster large exponential drop in tension to the relaxed state. Our aim is to see if recent insights into tension generation and the cross-bridge cycle can shed light on these kinetics (Davis and Epstein, 2009). In fully activated isometric rabbit psoas fibers two distinct and roughly equal populations of myosin heads are attached to actin. Half are termed “competent” and are capable of generating tension, while the other half are termed “noncompetent” and serve to “buffer” tension. For example, a step-decrease in fiber length releases a steric constraint on noncompetent cross-bridges thereby triggering tension recovery. We investigate whether the “buffer” of noncompetent AMD bridges might support the linear phase, with the exponential decline (kREL FAST) mediated by the decay of the remaining competent cross-bridge population. To do this, the linear phase was treated as a steady-state reaction with duration and not slope used to determine kREL SS. We found kREL SS normalized to kCAT (fiber ATPase) if a reasonable ∼21% of myosin heads constitute the noncompetent buffer. This leaves the final exponential decline to the relaxed state governed by the discharge of tension generating competent cross-bridges on exhaustion of the noncompetent buffer population. Additional support comes from the observations that added Pi decreases the duration and increases the slope of the linear phase with virtually no effect on kREL FAST (Tesi et al, 2002), and that a slow Tn Ca2+ off switch prolongs the linear phase (Kreutziger et al, 2008). Arrhenius plots of kCAT, kREL SS and kREL FAST are similar in slope, suggesting a common rate-limiting step of strain-sensitive ADP cross-bridge dissociation for all processes.

Kinetic Mechanism of Ca 2+-Induced Conformational Changes of Skeletal Muscle Troponin I in Rabbit Psoas Myofibrils

The kinetics of Ca2+-controlled conformational changes of the inhibitory subunit (sTnI) of the heterotrimeric skeletal troponin complex were determined by rapid stopped-flow at 10 °C. Conformational changes were probed by fluorescent labelling of sTnI at Cys134 located in between the second actin binding site of sTnI that interacts with actin-tropomyosin at low [Ca2+] and the switch peptide of sTnI that interacts with sTnC at high [Ca2+]. The kinetics was analysed for the sTn-complex in isolation and after its incorporation into rabbit psoas myofibrils. The rapid increase of [Ca2+] to pCa 4.5 induced biphasic fluorescence transients with similar rate constants of k+Ca1.phase ∼800 s−1 and k+Ca2.phase ∼100 s−1 in both preparations. Incorporation changed the polarity of the faster phase but not the polarity of the slower phase. Rapid reduction of [Ca2+] resulted in a monophasic fluorescence change whose rate constant k-Ca was 1.5 s−1 for isolated and 12 s−1 for incorporated sTn-complex. Thus, incorporation of the sTn-complex into the sarcomere increases the off-rate ∼8-fold while leaving the on-rate unaffected. The values of k+Ca2.phase and k-Ca determined in our myofibrils experiments are very similar to the values of kON and kOFF reported by Brenner & Chalovich (Biophys J. 1999 77:2692-708). They observed monophasic kinetics for sTnI labelled at Cys134 incorporated into skinned rabbit psoas fibers. In contrast to our experiments, they triggered sTnI-kinetics mechanically, i.e. by rapidly changing the number of force-generating cross-bridges. The faster conformational change in sTnI that is only observed in our Ca2+-triggered experiments is therefore likely associated with the Ca2+-binding process while the slower phase reports the conformational change of sTnI involved in force regulation. Supported by the Center of Molecular Medicine Cologne (CMMC-A6) and the DFG (SFB612-A2).

Response of Rigor Cross-bridges to Stretch Detected by Fluorescence Lifetime Imaging Microscopy of Myosin Essential Light Chain in Skeletal Muscle Fibers

We applied fluorescence lifetime imaging microscopy to map the microenvironment of the myosin essential light chain (ELC) in permeabilized skeletal muscle fibers. Four ELC mutants containing a single cysteine residue at different positions in the C-terminal half of the protein (ELC-127, ELC-142, ELC-160, and ELC-180) were generated by site-directed mutagenesis, labeled with 7-diethylamino-3-((((2-iodoacetamido)ethyl)amino)carbonyl)coumarin, and introduced into permeabilized rabbit psoas fibers. Binding to the myosin heavy chain was associated with a large conformational change in the ELC. When the fibers were moved from relaxation to rigor, the fluorescence lifetime increased for all label positions. However, when 1% stretch was applied to the rigor fibers, the lifetime decreased for ELC-127 and ELC-180 but did not change for ELC-142 and ELC-160. The differential change of fluorescence lifetime demonstrates the shift in position of the C-terminal domain of ELC with respect to the heavy chain and reveals specific locations in the lever arm region sensitive to the mechanical strain propagating from the actin-binding site to the lever arm.

Effect of Ca2+ binding properties of troponin C on rate of skeletal muscle force redevelopment

To investigate effects of altering troponin (Tn)C Ca(2+) binding properties on rate of skeletal muscle contraction, we generated three mutant TnCs with increased or decreased Ca(2+) sensitivities. Ca(2+) binding properties of the regulatory domain of TnC within the Tn complex were characterized by following the fluorescence of an IAANS probe attached onto the endogenous Cys(99) residue of TnC. Compared with IAANS-labeled wild-type Tn complex, V43QTnC, T70DTnC, and I60QTnC exhibited ∼1.9-fold higher, ∼5.0-fold lower, and ∼52-fold lower Ca(2+) sensitivity, respectively, and ∼3.6-fold slower, ∼5.7-fold faster, and ∼21-fold faster Ca(2+) dissociation rate (k(off)), respectively. On the basis of K(d) and k(off), these results suggest that the Ca(2+) association rate to the Tn complex decreased ∼2-fold for I60QTnC and V43QTnC. Constructs were reconstituted into single-skinned rabbit psoas fibers to assess Ca(2+) dependence of force development and rate of force redevelopment (k(tr)) at 15°C, resulting in sensitization of both force and k(tr) to Ca(2+) for V43QTnC, whereas T70DTnC and I60QTnC desensitized force and k(tr) to Ca(2+), I60QTnC causing a greater desensitization. In addition, T70DTnC and I60QTnC depressed both maximal force (F(max)) and maximal k(tr). Although V43QTnC and I60QTnC had drastically different effects on Ca(2+) binding properties of TnC, they both exhibited decreases in cooperativity of force production and elevated k(tr) at force levels <30%F(max) vs. wild-type TnC. However, at matched force levels >30%F(max) k(tr) was similar for all TnC constructs. These results suggest that the TnC mutants primarily affected k(tr) through modulating the level of thin filament activation and not by altering intrinsic cross-bridge cycling properties. To corroborate this, NEM-S1, a non-force-generating cross-bridge analog that activates the thin filament, fully recovered maximal k(tr) for I60QTnC at low Ca(2+) concentration. Thus TnC mutants with altered Ca(2+) binding properties can control the rate of contraction by modulating thin filament activation without directly affecting intrinsic cross-bridge cycling rates.

Interactions Between Connected Half-Sarcomeres Produce Emergent Mechanical Behavior in a Mathematical Model of Muscle

Most reductionist theories of muscle attribute a fiber's mechanical properties to the scaled behavior of a single half-sarcomere. Mathematical models of this type can explain many of the known mechanical properties of muscle but have to incorporate a passive mechanical component that becomes ∼300% stiffer in activating conditions to reproduce the force response elicited by stretching a fast mammalian muscle fiber. The available experimental data suggests that titin filaments, which are the mostly likely source of the passive component, become at most ∼30% stiffer in saturating Ca2+ solutions. The work described in this manuscript used computer modeling to test an alternative systems theory that attributes the stretch response of a mammalian fiber to the composite behavior of a collection of half-sarcomeres. The principal finding was that the stretch response of a chemically permeabilized rabbit psoas fiber could be reproduced with a framework consisting of 300 half-sarcomeres arranged in 6 parallel myofibrils without requiring titin filaments to stiffen in activating solutions. Ablation of inter-myofibrillar links in the computer simulations lowered isometric force values and lowered energy absorption during a stretch. This computed behavior mimics effects previously observed in experiments using muscles from desmin-deficient mice in which the connections between Z-disks in adjacent myofibrils are presumably compromised. The current simulations suggest that muscle fibers exhibit emergent properties that reflect interactions between half-sarcomeres and are not properties of a single half-sarcomere in isolation. It is therefore likely that full quantitative understanding of a fiber's mechanical properties requires detailed analysis of a complete fiber system and cannot be achieved by focusing solely on the properties of a single half-sarcomere.

The Effect of Glutathione on Skeletal Muscle Calcium Sensitivity and Myofilament Sulfhydryl Groups

Glutathione, a critical reducing agent present in relatively high levels(∼5mM) in skeletal muscle, can also attach to protein thiols via a disulfide bond. This process is referred to as glutathionylation, and is thought to be a protective mechanism to prevent irreversible protein oxidation. Prior studies have shown that when skinned fibers are exposed to reduced glutathione there is an increase in calcium sensitivity with no significant change in maximal force. These calcium sensitivity changes were largely reversible by the reducing agent DTT, indicating modification of protein thiols. We measured the force-pCa relationship of permeabilized rabbit psoas fibers treated with DTDP a thiol-specific oxidizing agent and glutathione (5mM). 2D gel electrophoresis using either IEF 4-6.5 or NEPHGE 3-10 in the first dimension was used to identify myofilament proteins whose sulfhydryl groups were modified with the oxidant-glutathione treatment. Additionally, phosphorylation of the regulatory myosin light chain was analyzed using 2d gels (IEF 4-6.5) and the phosphorylation specific stain Diamond Pro-Q. The pCa 50 of skinned psoas fibers was decreased upon exposure to DTT. Following DTT, the addition of DTDP and GSH sequentially, increased the calcium sensitivity. 2D gel electrophoresis, indicated myosin as a critical target for glutahionylation under these conditions. The RMLC was phosphorylated in the psoas fibers and the level remained constant during oxidant - glutathione treatment. In summary, our data suggest that glutathionylation of myofilament proteins can modulate calcium sensitivity, and may play an important role in maintaining muscle function during oxidative stress.

Effect of Inorganic Phosphate on the Rate of ADP Release During Ramp Shortening in Activated Permeabilized Fibers from Rabbit Psoas Muscle

A coumarin-labeled recombinant phosphorylated nucleoside diphosphate kinase (P∼NDPK-IDCC; West et al., 2009; Biophys.J. 96:3281-3294), was used as a fluorescence probe for time-resolved measurement of changes in [MgADP] during steady shortening of single permeabilized rabbit psoas fibers at 12°C (pCa 4.5, pH 7.1, ATP 5.7mM). A fiber contracted from the relaxed state by immersion into a Ca2+ activation solution at 0°C. Temperature activation was then initiated by immersion of the fiber into silicone oil at 12°C. The activation solutions were prepared with either zero added Pi or with 10 mM added Pi at constant ionic strength (0.15 M). The decline in fluorescence intensity emission (470nm) associated with MgADP-dependent dephosphorylation of P∼NDPK-IDCC (60 μM) was monitored during activation and during a period of isovelocity shortening. Fluorescence emission (580 nm) of a rhodamine dye was measured simultaneously to correct the P∼NDPK-IDCC signal for the effects of fiber movements and volume changes. The rate of MgADP release in the absence of added Pi increased from 0.7 ± 0.07 mM·s−1 at 0.2 fiber-lengths·s−1 to approximately 3.4 ± 0.25 mM·s−1 for shortening velocities between 1 and 2 fiber-lengths·s−1. When 10 mM Pi was added, the rate of ADP release at 0.2 fiber-lengths·s−1 was 0.48 ± 0.05 mM·s−1 and 2.6 ± 0.4 mM·s−1 at 1-2 fiber-lengths·s−1. In the absence of added Pi, the rate of ATP hydrolysis calculated from the appearance of ADP is similar to that calculated previously from the appearance of Pi using MDCC-labeled phosphate binding protein, over the same range of fiber shortening speeds (He et al., 1999, J. Physiol. 517: 839-854). Thus Pi slows the ATPase rate by 25-30%, both in the isometric and isotonic state. The energetic consequences will be discussed.

Interactions between Connected Half-Sarcomeres Produce Emergent Mechanical Behavior in a Mathematical Model of Muscle

Most reductionist theories of muscle attribute a fiber's mechanical properties to the scaled behavior of a single half-sarcomere. Mathematical models of this type can explain many of the known mechanical properties of muscle but have to incorporate a passive mechanical component that becomes ∼300% stiffer in activating conditions to reproduce the force response elicited by stretching a fast mammalian muscle fiber. The available experimental data suggests that titin filaments, which are the mostly likely source of the passive component, become at most ∼30% stiffer in saturating Ca2+ solutions. The work described in this manuscript used computer modeling to test an alternative systems theory that attributes the stretch response of a mammalian fiber to the composite behavior of a collection of half-sarcomeres. The principal finding was that the stretch response of a chemically permeabilized rabbit psoas fiber could be reproduced with a framework consisting of 300 half-sarcomeres arranged in 6 parallel myofibrils without requiring titin filaments to stiffen in activating solutions. Ablation of inter-myofibrillar links in the computer simulations lowered isometric force values and lowered energy absorption during a stretch. This computed behavior mimics effects previously observed in experiments using muscles from desmin-deficient mice in which the connections between Z-disks in adjacent myofibrils are presumably compromised. The current simulations suggest that muscle fibers exhibit emergent properties that reflect interactions between half-sarcomeres and are not properties of a single half-sarcomere in isolation. It is therefore likely that full quantitative understanding of a fiber's mechanical properties requires detailed analysis of a complete fiber system and cannot be achieved by focusing solely on the properties of a single half-sarcomere.

The Fast Skeletal Troponin Activator, CK‐2017357, Increases Skeletal Muscle Force in‐vitro and in‐situ

CK‐2017357 is a member of a class of fast skeletal troponin activators that were identified by high throughput screening of skeletal sarcomere preparations. We sought to understand how this compound altered force development in isometric muscle fibers. Treatment of skinned rabbit psoas fibers with CK‐2017357 caused a dose‐dependent shift of the force‐pCa relationship without altering the Hill slope or maximum force, consistent with a calcium sensitizing effect on force production. In living rat FDB muscle, CK‐2017357 (10 μM) increased sub‐tetanic force (150% of control at 10 Hz) without altering maximum force. Similar experiments were performed using rat EDL muscle in‐situ, where nervous and vascular connections were left intact. Infusions of 10 mg/kg increased sub‐tetanic force by 200% at 30 Hz. In summary, we have identified a skeletal troponin activator that sensitizes the sarcomere to calcium and results in increased sub‐maximal muscle force development in‐vitro and in‐situ. We believe that this may translate to improved muscle function in diseases where muscle is compromised due to injury, disease or age.

Time Course and Strain Dependence of ADP Release during Contraction of Permeabilized Skeletal Muscle Fibers

A phosphorylated, single cysteine mutant of nucleoside diphosphate kinase, labeled with N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide (P∼NDPK-IDCC), was used as a fluorescence probe for time-resolved measurement of changes in [MgADP] during contraction of single permeabilized rabbit psoas fibers. The dephosphorylation of the phosphorylated protein by MgADP occurs within the lattice environment of permeabilized fibers with a second-order rate constant at 12°C of 105 M−1 s−1. This dephosphorylation is accompanied by a change in coumarin fluorescence. We report the time course of P∼NDPK-IDCC dephosphorylation during the period of active isometric force redevelopment after quick release of fiber strain at pCa2+ of 4.5. After a rapid length decrease of 0.5% was applied to the fiber, the extra NDPK-IDCC produced during force recovery, above the value during the approximately steady state of isometric contraction, was 2.7 ± 0.6 μM and 4.7 ± 1.5 μM at 12 and 20°C, respectively. The rates of P∼NDPK-IDCC dephosphorylation during force recovery were 28 and 50 s−1 at 12 and 20°C, respectively. The time courses of isometric force and P∼NDPK-IDCC dephosphorylation were simulated using a seven-state reaction scheme. Relative isometric force was modeled by changes in the occupancy of strongly bound A.M.ADP.Pi and A.M.ADP states. A strain-sensitive A.M.ADP isomerization step was rate-limiting (3–6 s−1) in the cross-bridge turnover during isometric contraction. At 12°C, the A.M.ADP.Pi and the pre- and postisomerization A.M.ADP states comprised 56%, 38%, and 7% of the isometric force-bearing AM states, respectively. At 20°C, the force-bearing A.M.ADP.Pi state was a lower proportion of the total force-bearing states (37%), whereas the proportion of postisomerization A.M.ADP states was higher (19%). The simulations suggested that release of cross-bridge strain caused rapid depopulation of the preisomerization A.M.ADP state and transient accumulation of MgADP in the postisomerization A.M.ADP state. Hence, the strain-sensitive isomerization of A.M.ADP seems to explain the rate of change of P∼NDPK-IDCC dephosphorylation during force recovery. The temperature-dependent isometric distribution of myosin states is consistent with the previous observation of a small decrease in amplitude of the Pi transient during force recovery at 20°C and the current observation of an increase in amplitude of the ADP-sensitive NDPK-IDCC transient.

Structural Dynamics Of Myosin'S Light Chain Domain In A Pre-power Stroke Conformation

Cross-linking the two most reactive Cys of the myosin catalytic domain (CD) (SH1 and SH2) inhibits force production and ATP hydrolysis and locks myosin in a weak actin-binding conformation. Recent work using a bifunctional spin label (BSL) to crosslink SH1 and SH2 has shown that the CD is immobilized and orientationally disordered, suggesting that cross-linking traps myosin in a an intermediate state that primes the myosin head to generate force (Thompson et al., BJ, in press). In the present study, we measured light chain domain (LCD) structural dynamics in muscle fibers as a function of SH crosslinking. If the CD really is orientationally disordered by crosslinking and the two domains are structurally coupled, some of this disorder should be propagated to the LCD. To measure LCD structural dynamics, we used site-directed spin labeling to label chicken gizzard regulatory light chain (RLC), and then exchanged this labeled RLC for the native RLC in rabbit psoas fibers. Prior to crosslinking, EPR spectra acquired with the fiber axis parallel and perpendicular to the external field were very different, but cross-linking decreases this difference, indicating increased disorder. Saturation transfer EPR on these fibers showed that the heads remained immobile and thus attached to actin. These results support our hypothesis that SH1-SH2 crosslinking traps an actomyosin complex, possibly the first force-generating state in the power stroke, in which the CD is highly disordered and LCD is partially disordered, indicating a partially flexible linkage. A secondary goal of this research is to improve the technology for EPR on muscle fibers by developing a novel high-sensitivity EPR resonator for analysis of spin-label mobility, orientation, and force on labeled fibers.