Myofilament length-dependent activation develops within 5 ms in guinea-pig myocardium

Myofilament length-dependent activation is a universal property of striated muscle, yet the molecular mechanisms that underlie this phenomenon are incompletely understood. Additionally, the rate by which sarcomere length (SL) is sensed and then transduced to form length-dependent activation is unknown. Here, using isolated guinea-pig myocardium, we employed a rapid solution-switch single myofibril technique that allows for the study of contractile action/relaxation dynamics in the virtual absence of diffusion delays. We compared contraction kinetics obtained at submaximal activation at steady-state SL with contractions observed after rapid SL ramps to that same SL just before activation. Neither the activation and relaxation kinetics nor the final submaximal force development differed significantly between the two contraction modes for SL ramps as fast as 5 ms. We conclude that the transduction of the length signal by the cardiac sarcomere to modulate thin filament activation levels occurs virtually instantaneously, possibly resulting from structural rearrangements of the contractile proteins.     

Force recovery after activated shortening in whole skeletal muscle: transient and steady-state aspects of force depression.

The depression of isometric force after active shortening is a well-accepted characteristic of skeletal muscle, yet its mechanisms remain unknown. Although traditionally analyzed at steady state, transient phenomena caused, at least in part, by cross-bridge kinetics may provide novel insight into the mechanisms associated with force depression (FD). To identify the transient aspects of FD and its relation to shortening speed, shortening amplitude, and muscle mechanical work, in situ experiments were conducted in soleus muscle-tendon units of anesthetized cats. The period immediately after shortening, in which force recovers toward steady state, was fit by using an exponential recovery function (R2 > 0.99). Statistical analyses revealed that steady-state FD (FD(ss)) increased with shortening amplitude and mechanical work. This FD(ss) increase was always accompanied by a significant decrease in force recovery rate. Furthermore, a significant reduction in stiffness was observed after all activated shortenings, presumably because of a reduced proportion of attached cross bridges. These results were interpreted with respect to the two most prominent proposed mechanisms of force depression: sarcomere length nonuniformity theory (7, 32) and a stress-induced inhibition of cross-bridge binding in the newly formed actin-myosin overlap zone (14, 28). We hypothesized that the latter could describe both steady-state and transient aspects of FD using a single scalar variable, the mechanical work done during shortening. As either excursion (overlap) or force (stress) is increased, mechanical work increases, and cross-bridge attachment would become more inhibited, as supported by this study in which an increase in mechanical work resulted in a slower recovery to a more depressed steady-state force.     

Modelling concentric contraction of muscle using an improved cross-bridge model.

Despite its overwhelming acceptance in muscle research, the cross-bridge theory does not account for all phenomena observed during muscular contractions. A phenomenon which has received much attention in the biomechanics literature, but has evaded convincing explanation and is not accounted for in the formulation of the classic cross-bridge theory, is the persistent aftereffects of muscular length changes on force production. For example, following muscle shortening, the isometric force of a muscle is depressed for a long time period ( > 5 s) compared to the corresponding isometric force following no length change. In the present study, the classic cross-bridge model was modified in two ways in an attempt to account for the force depressions following muscle shortening. First, the steady-state force depressions following shortening were described by a single scalar variable: the work performed by the muscle during shortening; and second, the dynamic, history-dependent cross-bridge properties were described using a fading memory function. The proposed model was developed and tested for shortening of the cat soleus at constant speeds ranging from 4 to 32 mm/s, for shortening at changing speeds, and for shortening of different magnitudes ranging from 2 to 10 mm. The history-dependent forces during shortening and the steady-state force depressions following shortening were well captured with the modified cross-bridge model. The present model contains two mathematically simple adaptations to the classic cross-bridge model, and is the first such model to account for the long-lasting force depressions following muscle shortening using a single scalar variable.     

Compliant realignment of binding sites in muscle: transient behavior and mechanical tuning.

The presence of compliance in the lattice of filaments in muscle raises a number of concerns about how one accounts for force generation in the context of the cross-bridge cycle--binding site motions and coupling between cross-bridges confound more traditional analyses. To explore these issues, we developed a spatially explicit, mechanochemical model of skeletal muscle contraction. With a simple three-state model of the cross-bridge cycle, we used a Monte Carlo simulation to compute the instantaneous balance of forces throughout the filament lattice, accounting for both thin and thick filament distortions in response to cross-bridge forces. This approach is compared to more traditional mass action kinetic models (in the form of coupled partial differential equations) that assume filament inextensibility. We also monitored instantaneous force generation, ATP utilization, and the dynamics of the cross-bridge cycle in simulations of step changes in length and variations in shortening velocity. Three critical results emerge from our analyses: 1) there is a significant realignment of actin-binding sites in response to cross-bridge forces, 2) this realignment recruits additional cross-bridge binding, and 3) we predict mechanical behaviors that are consistent with experimental results for velocity and length transients. Binding site realignment depends on the relative compliance of the filament lattice and cross-bridges, and within the measured range of these parameters, gives rise to a sharply tuned peak for force generation. Such mechanical tuning at the molecular level is the result of mechanical coupling between individual cross-bridges, mediated by thick filament deformations, and the resultant realignment of binding sites on the thin filament.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Perturbed equilibria of myosin binding in airway smooth muscle: bond-length distributions, mechanics, and ATP metabolism

We carried out a detailed mathematical analysis of the effects of length fluctuations on the dynamically evolving cross-bridge distributions, simulating those that occur in airway smooth muscle during breathing. We used the latch regulation scheme of Hai and Murphy (Am. J. Physiol. Cell Physiol. 255:C86-C94, 1988) integrated with Huxley's sliding filament theory of muscle contraction. This analysis showed that imposed length fluctuations decrease the mean number of attached bridges, depress muscle force and stiffness, and increase force-length hysteresis. At frequencies >0.1 Hz, the bond-length distribution of slowly cycling latch bridges changed little over the stretch cycle and contributed almost elastically to muscle force, but the rapidly cycling cross-bridge distribution changed substantially and dominated the hysteresis. By contrast, at frequencies <0.033 Hz this behavior was reversed: the rapid cycling cross-bridge distribution changed little, effectively functioning as a constant force generator, while the latch bridge bond distribution changed substantially and dominated the stiffness and hysteresis. The analysis showed the dissociation of force/length hysteresis and cross-bridge cycling rates when strain amplitude exceeds 3%; that is, there is only a weak coupling between net external mechanical work and the ATP consumption required for cycling cross-bridges during the oscillatory steady state. Although these results are specific to airway smooth muscle, the approach generalizes to other smooth muscles subjected to cyclic length fluctuations.     

A theory on auto-oscillation and contraction in striated muscle

It is widely accepted that muscle cells take either force-generating or relaxing state in an all-or-none fashion through the so-called excitation–contraction coupling. On the other hand, the membrane-less contractile apparatus takes the third state, i.e., the auto-oscillation (SPOC) state, at the activation level that is intermediate between full activation and relaxation. Here, to explain the dynamics of all three states of muscle, we construct a novel theoretical model based on the balance of forces not only parallel but also perpendicular to the long axis of myofibrils, taking into account the experimental fact that the spacing of myofilament lattice changes with sarcomere length and upon contraction. This theory presents a phase diagram composed of several states of the contractile apparatus and explains the dynamic behavior of SPOC, e.g., periodical changes in sarcomere length with the saw-tooth waveform. The appropriate selection of the constant of the molecular friction due to the cross-bridge formation can explain the difference in the SPOC periods observed under various activating conditions and in different muscle types, i.e., skeletal and cardiac. The theory also predicts the existence of a weak oscillation state at the boundary between SPOC and relaxation regions in the phase diagram. Thus, the present theory comprehensively explains the characteristics of auto-oscillation and contraction in the contractile system of striated muscle.     

Cross-bridge induced force enhancement?

When a muscle is stretched while activated, its steady-state isometric force following stretch is greater than the corresponding purely isometric force. This so-called residual force enhancement (RFE) has been observed for half a century, yet its mechanism remains unknown. Recent experiments suggest that RFE is not caused by non-uniformities in sarcomere lengths, as had been assumed for a long time, and cannot be explained primarily with increases in passive force, but is directly related to the kinetics of the cross-bridge cycle. Specifically, it has been suggested that stretching an attached cross-bridge increases its dwell time and duty ratio; therefore, the proportion of attached cross-bridges in a muscle would be increased by stretch, thereby causing RFE. A three bead laser trap setup was used for testing single cross-bridge (myosin II) interactions with actin. Upon attachment of a cross-bridge, a stretch or shortening of the cross-bridge was applied with a force of about 1.0 pN. The hypothesis that stretching a single cross-bridge increases its dwell time and duty ratio was rejected. However, stretching caused an increase in the average steady-state force per cross-bridge (3.4±0.4 pN; n=433) compared to shortening (1.9±0.3 pN; n=689). Therefore, based on the results of this study, RFE cannot be explained by an increased duty ratio and the associated increase in proportion of attached cross-bridges, but might be associated with an increased force per cross-bridge.     

Sarcomere Length Nonuniformity and Force Regulation in Myofibrils and Sarcomeres

The smallest contractile unit in striated muscles is the sarcomere. Although some of the classic features of contraction assume a uniform behavior of sarcomeres within myofibrils, the occurrence of sarcomere length nonuniformities has been well recognized for years, but it is yet not well understood. In the past years, there has been a great advance in experiments using isolated myofibrils and sarcomeres that has allowed scientists to directly evaluate sarcomere length nonuniformity. This review will focus on studies conducted with these preparations to develop the hypotheses that 1) force production in myofibrils is largely altered and regulated by intersarcomere dynamics and that 2) the mechanical work of one sarcomere in a myofibril is transmitted to other sarcomeres in series. We evaluated studies looking into myofibril activation, relaxation, and force changes produced during activation. We conclude that force production in myofibrils is largely regulated by intersarcomere dynamics, which arises from the cooperative work of the contractile and elastic elements within a myofibril.     

An expanded latch-bridge model of protein kinase C-mediated smooth muscle contraction.

A thin-filament-regulated latch-bridge model of smooth muscle contraction is proposed to integrate thin-filament-based inhibition of actomyosin ATPase activity with myosin phosphorylation in the regulation of smooth muscle mechanics. The model included two latch-bridge cycles, one of which was identical to the four-state model as proposed by Hai and Murphy (Am J Physiol Cell Physiol 255: C86-C94, 1988), whereas the ultraslow cross-bridge cycle has lower cross-bridge cycling rates. The model-fitted phorbol ester induced slow contractions at constant myosin phosphorylation and predicted steeper dependence of force on myosin phosphorylation in phorbol ester-stimulated smooth muscle. By shifting cross bridges between the two latch-bridge cycles, the model predicts that a smooth muscle cell can either maintain force at extremely low-energy cost or change its contractile state rapidly, if necessary. Depending on the fraction of cross bridges engaged in the ultraslow latch-bridge cycle, the model predicted biphasic kinetics of smooth muscle mechanics and variable steady-state dependencies of force and shortening velocity on myosin phosphorylation. These results suggest that thin-filament-based regulatory proteins may function as tuners of actomyosin ATPase activity, thus allowing a smooth muscle cell to have two discrete cross-bridge cycles with different cross-bridge cycling rates.     

The smooth muscle cross-bridge cycle studied using sinusoidal length perturbations

The mechanical characteristics of smooth muscle can be broadly defined as either phasic, or fast contracting, and tonic, or slow contracting (, Pharmacol. Rev. 20:197-272). To determine if differences in the cross-bridge cycle and/or distribution of the cross-bridge states could contribute to differences in the mechanical properties of smooth muscle, we determined force and stiffness as a function of frequency in Triton-permeabilized strips of rabbit portal vein (phasic) and aorta (tonic). Permeabilized muscle strips were mounted between a piezoelectric length driver and a piezoresistive force transducer. Muscle length was oscillated from 1 to 100 Hz, and the stiffness was determined as a function of frequency from the resulting force response. During calcium activation (pCa 4, 5 mM MgATP), force and stiffness increased to steady-state levels consistent with the attachment of actively cycling cross-bridges. In smooth muscle, because the cross-bridge states involved in force production have yet to be elucidated, the effects of elevation of inorganic phosphate (P(i)) and MgADP on steady-state force and stiffness were examined. When portal vein strips were transferred from activating solution (pCa 4, 5 mM MgATP) to activating solution with 12 mM P(i), force and stiffness decreased proportionally, suggesting that cross-bridge attachment is associated with P(i) release. For the aorta, elevating P(i) decreased force more than stiffness, suggesting the existence of an attached, low-force actin-myosin-ADP- P(i) state. When portal vein strips were transferred from activating solution (pCa 4, 5 mM MgATP) to activating solution with 5 mM MgADP, force remained relatively constant, while stiffness decreased approximately 50%. For the aorta, elevating MgADP decreased force and stiffness proportionally, suggesting for tonic smooth muscle that a significant portion of force production is associated with ADP release. These data suggest that in the portal vein, force is produced either concurrently with or after P(i) release but before MgADP release, whereas in aorta, MgADP release is associated with a portion of the cross-bridge powerstroke. These differences in cross-bridge properties could contribute to the mechanical differences in properties of phasic and tonic smooth muscle.     

Cross-bridge versus thin filament contributions to the level and rate of force development in cardiac muscle

In striated muscle thin filament activation is initiated by Ca(2+) binding to troponin C and augmented by strong myosin binding to actin (cross-bridge formation). Several lines of evidence have led us to hypothesize that thin filament properties may limit the level and rate of force development in cardiac muscle at all levels of Ca(2+) activation. As a test of this hypothesis we varied the cross-bridge contribution to thin filament activation by substituting 2 deoxy-ATP (dATP; a strong cross-bridge augmenter) for ATP as the contractile substrate and compared steady-state force and stiffness, and the rate of force redevelopment (k(tr)) in demembranated rat cardiac trabeculae as [Ca(2+)] was varied. We also tested whether thin filament dynamics limits force development kinetics during maximal Ca(2+) activation by comparing the rate of force development (k(Ca)) after a step increase in [Ca(2+)] with photorelease of Ca(2+) from NP-EGTA to maximal k(tr), where Ca(2+) binding to thin filaments should be in (near) equilibrium during force redevelopment. dATP enhanced steady-state force and stiffness at all levels of Ca(2+) activation. At similar submaximal levels of steady-state force there was no increase in k(tr) with dATP, but k(tr) was enhanced at higher Ca(2+) concentrations, resulting in an extension (not elevation) of the k(tr)-force relationship. Interestingly, we found that maximal k(tr) was faster than k(Ca), and that dATP increased both by a similar amount. Our data suggest the dynamics of Ca(2+)-mediated thin filament activation limits the rate that force develops in rat cardiac muscle, even at saturating levels of Ca(2+).     

Computer simulation of movement-generating cross-bridges.

A stochastic computational method was developed to study properties of cross-bridge models for muscle contraction, by following the time history of individual cross-bridge model of Andrew Huxley (1957) and a modified two-state model with more realistic behavior during steady stretching are used as examples. The method can readily compute steady-state force during shortening and stretching and force-transients following rapid changes in length. Computations of velocity with a steady load and of velocity transients are more sensitive to the randomness inherent in the stochastic method.     

Thixotropy and Rheopexy of Muscle Fibers Probed Using Sinusoidal Oscillations

Length changes of muscle fibers have previously been shown to result in a temporary reduction in fiber stiffness that is referred to as thixotropy. Understanding the mechanism of this thixotropy is important to our understanding of muscle function since there are many instances in which muscle is subjected to repeated patterns of lengthening and shortening. By applying sinusoidal length changes to one end of single permeabilized muscle fibers and measuring the force response at the opposite end, we studied the history-dependent stiffness of both relaxed and activated muscle fibers. For length change oscillations greater than 1 Hz, we observed thixotropic behavior of activated fibers. Treatment of these fibers with EDTA and blebbistatin, which inhibits myosin-actin interactions, quashed this effect, suggesting that the mechanism of muscle fiber thixotropy is cross-bridge dependent. We modeled a half-sarcomere experiencing sinusoidal length changes, and our simulations suggest that thixotropy could arise from force-dependent cross-bridge kinetics. Surprisingly, we also observed that, for length change oscillations less than 1 Hz, the muscle fiber exhibited rheopexy. In other words, the stiffness of the fiber increased in response to the length changes. Blebbistatin and EDTA did not disrupt the rheopectic behavior, suggesting that a non-cross-bridge mechanism contributes to this phenomenon.     

The mechanism of muscle contraction

Knowledge of the mechanism of contraction has been obtained from studies of the interaction of actin and myosin in solution, from an elucidation of the structure of muscle fibers, and from measurements of the mechanics and energetics of fiber contraction. Many of the states and the transition rates between them have been established for the hydrolysis of ATP by actin and myosin subfragments in solution. A major goal is to now understand how the kinetics of this interaction are altered when it occurs in the organized array of the myofibril. Early work on the structure of muscle suggested that changes in the orientation of myosin cross-bridges were responsible for the generation of force. More recently, fluorescent and paramagnetic probes attached to the cross-bridges have suggested that at least some domains of the cross-bridges do not change orientation during force generation. A number of properties of active cross-bridges have been defined by measurements of steady state contractions of fibers and by the transients which follow step changes in fiber length or tension. Taken together these studies have provided firm evidence that force is generated by a cyclic interaction in which a myosin cross-bridge attaches to actin, exerts force through a "powerstroke" of 12 nm, and is then released by the binding of ATP. The mechanism of this interaction at the molecular level remains unknown.     

Tissue length modulates "stimulated actin polymerization," force augmentation, and the rate of swine carotid arterial contraction

"Stimulated actin polymerization" has been proposed to be involved in force augmentation, in which prior submaximal activation of vascular smooth muscle increases the force of a subsequent maximal contraction by ～15%. In this study, we altered stimulated actin polymerization by adjusting tissue length and then measured the effect on force augmentation. At optimal tissue length (1.0 L(o)), force augmentation was observed and was associated with increased prior stimulated actin polymerization, as evidenced by increased prior Y118 paxillin phosphorylation without changes in prior S3 cofilin or cross-bridge phosphorylation. Tissue length, per se, regulated Y118 paxillin, but not S3 cofilin, phosphorylation. At short tissue length (0.6 L(o)), force augmentation was observed and was associated with increased prior stimulated actin polymerization, as evidenced by reduced prior S3 cofilin phosphorylation without changes in Y118 paxillin or cross-bridge phosphorylation. At long tissue length (1.4 L(o)), force augmentation was not observed, and there were no prior changes in Y118 paxillin, S3 cofilin, or cross-bridge phosphorylation. There were no significant differences in the cross-bridge phosphorylation transients before and after the force augmentation protocol at all three lengths tested. Tissues contracted faster at longer tissue lengths; contractile rate correlated with prior Y118 paxillin phosphorylation. Total stress, per se, predicted Y118 paxillin phosphorylation. These data suggest that force augmentation is regulated by stimulated actin polymerization and that stimulated actin polymerization is regulated by total arterial stress. We suggest that K(+) depolarization first leads to cross-bridge phosphorylation and contraction, and the contraction-induced increase in mechanical strain increases Y118 paxillin phosphorylation, leading to stimulated actin polymerization, which further increases force, i.e., force augmentation and, possibly, latch.     

Molecular Characterisation of Titin N2A and Its Binding of CARP Reveals a Titin/Actin Cross-linking Mechanism

Striated muscle responds to mechanical overload by rapidly up-regulating the expression of the cardiac ankyrin repeat protein, CARP, which then targets the sarcomere by binding to titin N2A in the I-band region. To date, the role of this interaction in the stress response of muscle remains poorly understood. Here, we characterise the molecular structure of the CARP-receptor site in titin (UN2A) and its binding of CARP. We find that titin UN2A contains a central three-helix bundle fold (ca 45 residues in length) that is joined to N- and C-terminal flanking immunoglobulin domains by long, flexible linkers with partial helical content. CARP binds titin by engaging an α-hairpin in the three-helix fold of UN2A, the C-terminal linker sequence, and the BC loop in Ig81, which jointly form a broad binding interface. Mutagenesis showed that the CARP/N2A association withstands sequence variations in titin N2A and we use this information to evaluate 85 human single nucleotide variants. In addition, actin co-sedimentation, co-transfection in C2C12 cells, proteomics on heart lysates, and the mechanical response of CARP-soaked myofibrils imply that CARP induces the cross-linking of titin and actin myofilaments, thereby increasing myofibril stiffness. We conclude that CARP acts as a regulator of force output in the sarcomere that preserves muscle mechanical performance upon overload stress.     

Mysteries of muscle contraction

According to the cross-bridge theory, the steady-state isometric force of a muscle is given by the amount of actin-myosin filament overlap. However, it has been known for more than half a century that steady-state forces depend crucially on contractile history. Here, we examine history-dependent steady-state force production in view of the cross-bridge theory, available experimental evidence, and existing explanations for this phenomenon. This is done on various structural levels, ranging from the intact muscle to the myofibrillar and isolated contractile protein level, so that advantages and limitations of the various preparations can be fully exploited and overcome. Based on experimental evidence, we conclude that steady-state force following active muscle stretching is enhanced, and this enhancement has a passive and an active component. The active component is associated with the cross-bridge kinetics, and the passive component is associated with a calcium-dependent increase in titin stiffness.     

Muscle anatomy is a primary determinant of muscle relaxation dynamics in the lobster (<Emphasis Type="Italic">Panulirus interruptus</Emphasis>) stomatogastric system

We stained sarcomere thin filaments with fluorescently labeled phalloidin, measured sarcomere and muscle length, and calculated sarcomere number in pyloric and gastric mill muscles. A wide range of sarcomere lengths (3.25–12.29 μm), muscle lengths (5.9–21.1 mm), and sarcomere numbers (648–3,036) were observed. Sarcomere number differences occurred both because of changes in sarcomere length and muscle length, and sarcomere and muscle length varied independently. This independence, the wide range of sarcomere numbers present, and the muscles being all ‘slow’, graded muscles allowed us to use these data to test Huxley and Neidergerke’s (1954) hypothesis that muscle dynamics depend on sarcomere number. The time constants of exponential fits to contraction relaxations were used to measure muscle dynamics, and comparison of theoretical predictions and experimental results quantitatively confirm the predicted dependence. The differing dynamics of the various pyloric muscles are likely functionally important, and the dependence of muscle dynamics on sarcomere number implies that sarcomere number is likely closely regulated in these muscles. The stomatogastric system may thus be an excellent model system for studying the mechanisms regulating muscle sarcomere number.     

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.