Omecamtiv Mecarbil Slows Myosin Kinetics in Skinned Rat Myocardium at Physiological Temperature

Heart failure is a life-threatening condition that occurs when the heart muscle becomes weakened and cannot adequately circulate blood and nutrients around the body. Omecamtiv mecarbil (OM) is a compound that has been developed to treat systolic heart failure via targeting the cardiac myosin heavy chain to increase myocardial contractility. Biophysical and biochemical studies have found that OM increases calcium (Ca²⁺) sensitivity of contraction by prolonging the myosin working stroke and increasing the actin-myosin cross-bridge duty ratio. Most in vitro studies probing the effects of OM on cross-bridge kinetics and muscle force production have been conducted at subphysiological temperature, even though temperature plays a critical role in enzyme activity and cross-bridge function. Herein, we used skinned, ventricular papillary muscle strips from rats to investigate the effects of [OM] on Ca²⁺-activated force production, cross-bridge kinetics, and myocardial viscoelasticity at physiological temperature (37°C). We find that OM only increases myocardial contractility at submaximal Ca²⁺ activation levels and not maximal Ca²⁺ activation levels. As [OM] increased, the kinetic rate constants for cross-bridge recruitment and detachment slowed for both submaximal and maximal Ca²⁺-activated conditions. These findings support a mechanism by which OM increases cardiac contractility at physiological temperature via increasing cross-bridge contributions to thin-filament activation as cross-bridge kinetics slow and the duration of cross-bridge attachment increases. Thus, force only increases at submaximal Ca²⁺ activation due to cooperative recruitment of neighboring cross-bridges, because thin-filament activation is not already saturated. In contrast, OM does not increase myocardial force production for maximal Ca²⁺-activated conditions at physiological temperature because cooperative activation of thin filaments may already be saturated.     

Sarcomere lattice geometry influences cooperative myosin binding in muscle

In muscle, force emerges from myosin binding with actin (forming a cross-bridge). This actomyosin binding depends upon myofilament geometry, kinetics of thin-filament Ca²⁺ activation, and kinetics of cross-bridge cycling. Binding occurs within a compliant network of protein filaments where there is mechanical coupling between myosins along the thick-filament backbone and between actin monomers along the thin filament. Such mechanical coupling precludes using ordinary differential equation models when examining the effects of lattice geometry, kinetics, or compliance on force production. This study uses two stochastically driven, spatially explicit models to predict levels of cross-bridge binding, force, thin-filament Ca²⁺ activation, and ATP utilization. One model incorporates the 2-to-1 ratio of thin to thick filaments of vertebrate striated muscle (multi-filament model), while the other comprises only one thick and one thin filament (two-filament model). Simulations comparing these models show that the multi-filament predictions of force, fractional cross-bridge binding, and cross-bridge turnover are more consistent with published experimental values. Furthermore, the values predicted by the multi-filament model are greater than those values predicted by the two-filament model. These increases are larger than the relative increase of potential inter-filament interactions in the multi-filament model versus the two-filament model. This amplification of coordinated cross-bridge binding and cycling indicates a mechanism of cooperativity that depends on sarcomere lattice geometry, specifically the ratio and arrangement of myofilaments.     

Filament compliance effects can explain tension overshoots during force development

Spatially explicit stochastic simulations of myosin S1 heads attaching to a single actin filament were used to investigate the process of force development in contracting muscle. Filament compliance effects were incorporated by adjusting the spacing between adjacent actin binding sites and adjacent myosin heads in response to cross-bridge attachment/detachment events. Appropriate model parameters were determined by multi-dimensional optimization and used to simulate force development records corresponding to different levels of Ca(2+) activation. Simulations in which the spacing between both adjacent actin binding sites and adjacent myosin S1 heads changed by approximately 0.06 nm after cross-bridge attachment/detachment events 1), exhibited tension overshoots with a Ca(2+) dependence similar to that measured experimentally and 2), mimicked the observed k(tr)-relative tension relationship without invoking a Ca(2+)-dependent increase in the rate of cross-bridge state transitions. Tension did not overshoot its steady-state value in control simulations modeling rigid thick and thin filaments with otherwise identical parameters. These results underline the importance of filament geometry and actin binding site availability in quantitative theories of muscle contraction.     

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.     

Cooperative attachment of cross bridges predicts regulation of smooth muscle force by myosin phosphorylation

Serine 19 phosphorylation of the myosin regulatory light chain (MRLC) appears to be the primary determinant of smooth muscle force development. The relationship between MRLC phosphorylation and force is nonlinear, showing that phosphorylation is not a simple switch regulating the number of cycling cross bridges. We reexamined the MRLC phosphorylation-force relationship in slow, tonic swine carotid media; fast, phasic rabbit urinary bladder detrusor; and very fast, tonic rat anococcygeus. We found a sigmoidal dependence of force on MRLC phosphorylation in all three tissues with a threshold for force development of approximately 0.15 mol P(i)/mol MRLC. This behavior suggests that force is regulated in a highly cooperative manner. We then determined whether a model that employs both the latch-bridge hypothesis and cooperative activation could reproduce the relationship between Ser(19)-MRLC phosphorylation and force without the need for a second regulatory system. We based this model on skeletal muscle in which attached cross bridges cooperatively activate thin filaments to facilitate cross-bridge attachment. We found that such a model describes both the steady-state and time-course relationship between Ser(19)-MRLC phosphorylation and force. The model required both cooperative activation and latch-bridge formation to predict force. The best fit of the model occurred when binding of a cross bridge cooperatively activated seven myosin binding sites on the thin filament. This result suggests cooperative mechanisms analogous to skeletal muscle that will require testing.     

Coupling of Adjacent Tropomyosins Enhances Cross-Bridge-Mediated Cooperative Activation in a Markov Model of the Cardiac Thin Filament

We developed a Markov model of cardiac thin filament activation that accounts for interactions among nearest-neighbor regulatory units (RUs) in a spatially explicit manner. Interactions were assumed to arise from structural coupling of adjacent tropomyosins (Tms), such that Tm shifting within each RU was influenced by the Tm status of its neighbors. Simulations using the model demonstrate that this coupling is sufficient to produce observed cooperativity in both steady-state and dynamic force-Ca²⁺ relationships. The model was further validated by comparison with reported responses under various conditions including inhibition of myosin binding and the addition of strong-binding, non-force-producing myosin fragments. The model also reproduced the effects of 2.5 mM added P_i on Ca²⁺-activated force and the rate of force redevelopment measured in skinned rat myocardial preparations. Model analysis suggests that Tm-Tm coupling potentiates the activating effects of strongly-bound cross-bridges and contributes to force-Ca²⁺ dynamics of intact cardiac muscle. The model further predicts that activation at low Ca²⁺ concentrations is cooperatively inhibited by nearest neighbors, requiring Ca²⁺ binding to >25% of RUs to produce appreciable levels of force. Without excluding other putative cooperative mechanisms, these findings suggest that structural coupling of adjacent Tm molecules contributes to several properties of cardiac myofilament activation.     

Millisecond time-resolved changes occurring in Ca2+-regulated myosin filaments upon relaxation

Contraction of many muscles is activated in part by the binding of Ca²⁺ to, or phosphorylation of, the myosin heads on the surface of the thick filaments. In relaxed muscle, the myosin heads are helically ordered and undergo minimal interaction with actin. On Ca²⁺ binding or phosphorylation, the head array becomes disordered, reflecting breakage of the head-head and other interactions that underlie the ordered structure. Loosening of the heads from the filament surface enables them to interact with actin filaments, bringing about contraction. On relaxation, the heads return to their ordered positions on the filament backbone. In scallop striated adductor muscle, the disordering that takes place on Ca²⁺ binding occurs on the millisecond time scale, suggesting that it is a key element of muscle activation. Here we have studied the reverse process. Using time-resolved negative staining electron microscopy, we show that the rate of reordering on removal of Ca²⁺ also occurs on the same physiological time scale. Direct observation of images together with analysis of their Fourier transforms shows that activated heads regain their axial ordering within 20 ms and become ordered in their final helical positions within 50 ms. This rapid reordering suggests that reformation of the ordered structure, and the head-head and other interactions that underlie it, is a critical element of the relaxation process.     

Cooperative mechanisms in the activation dependence of the rate of force development in rabbit skinned skeletal muscle fibers

Regulation of contraction in skeletal muscle is a highly cooperative process involving Ca(2+) binding to troponin C (TnC) and strong binding of myosin cross-bridges to actin. To further investigate the role(s) of cooperation in activating the kinetics of cross-bridge cycling, we measured the Ca(2+) dependence of the rate constant of force redevelopment (k(tr)) in skinned single fibers in which cross-bridge and Ca(2+) binding were also perturbed. Ca(2+) sensitivity of tension, the steepness of the force-pCa relationship, and Ca(2+) dependence of k(tr) were measured in skinned fibers that were (1) treated with NEM-S1, a strong-binding, non-force-generating derivative of myosin subfragment 1, to promote cooperative strong binding of endogenous cross-bridges to actin; (2) subjected to partial extraction of TnC to disrupt the spread of activation along the thin filament; or (3) both, partial extraction of TnC and treatment with NEM-S1. The steepness of the force-pCa relationship was consistently reduced by treatment with NEM-S1, by partial extraction of TnC, or by a combination of TnC extraction and NEM-S1, indicating a decrease in the apparent cooperativity of activation. Partial extraction of TnC or NEM-S1 treatment accelerated the rate of force redevelopment at each submaximal force, but had no effect on kinetics of force development in maximally activated preparations. At low levels of Ca(2+), 3 microM NEM-S1 increased k(tr) to maximal values, and higher concentrations of NEM-S1 (6 or 10 microM) increased k(tr) to greater than maximal values. NEM-S1 also accelerated k(tr) at intermediate levels of activation, but to values that were submaximal. However, the combination of partial TnC extraction and 6 microM NEM-S1 increased k(tr) to virtually identical supramaximal values at all levels of activation, thus, completely eliminating the activation dependence of k(tr). These results show that k(tr) is not maximal in control fibers, even at saturating [Ca(2+)], and suggest that activation dependence of k(tr) is due to the combined activating effects of Ca(2+) binding to TnC and cross-bridge binding to actin.     

A molecular model of phosphorylation-based activation and potentiation of tarantula muscle thick filaments

Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). To elucidate the structural mechanism of activation, we have studied RLC phosphorylation in tarantula thick filaments, whose high-resolution structure is known. In the relaxed state, tarantula RLCs are ∼ 50% non-phosphorylated and 50% mono-phosphorylated, while on activation, mono-phosphorylation increases, and some RLCs become bi-phosphorylated. Mass spectrometry shows that relaxed-state mono-phosphorylation occurs on Ser35, while Ca²⁺-activated phosphorylation is on Ser45, both located near the RLC N-terminus. The sequences around these serines suggest that they are the targets for protein kinase C and myosin light chain kinase (MLCK), respectively. The atomic model of the tarantula filament shows that the two myosin heads (“free” and “blocked”) are in different environments, with only the free head serines readily accessible to kinases. Thus, protein kinase C Ser35 mono-phosphorylation in relaxed filaments would occur only on the free heads. Structural considerations suggest that these heads are less strongly bound to the filament backbone and may oscillate occasionally between attached and detached states (“swaying” heads). These heads would be available for immediate actin interaction upon Ca²⁺ activation of the thin filaments. Once MLCK becomes activated, it phosphorylates free heads on Ser45. These heads become fully mobile, exposing blocked head Ser45 to MLCK. This would release the blocked heads, allowing their interaction with actin. On this model, twitch force would be produced by rapid interaction of swaying free heads with activated thin filaments, while prolonged exposure to Ca²⁺ on tetanus would recruit new MLCK-activated heads, resulting in force potentiation.     

Altered kinetics of contraction in skeletal muscle fibers containing a mutant myosin regulatory light chain with reduced divalent cation binding.

We examined the kinetic properties of rabbit skinned skeletal muscle fibers in which the endogenous myosin regulatory light chain (RLC) was partially replaced with a mutant RLC (D47A) containing a point mutation within the Ca2+/Mg2+ binding site that severely reduced its affinity for divalent cations. We found that when approximately 50% of the endogenous RLC was replaced by the mutant, maximum tension declined to approximately 60% of control and the rate constant of active tension redevelopment (ktr) after mechanical disruption of cross-bridges was reduced to approximately 70% of control. This reduction in ktr was not an indirect effect on kinetics due to a reduced number of strongly bound myosin heads, because when the strongly binding cross-bridge analog N-ethylmaleimide-modified myosin subfragment1 (NEM-S1) was added to the fibers, there was no effect upon maximum ktr. Fiber stiffness declined after D47A exchange in a manner indicative of a decrease in the number of strongly bound cross-bridges, suggesting that the force per cross-bridge was not significantly affected by the presence of D47A RLC. In contrast to the effects on ktr, the rate of tension relaxation in steadily activated fibers after flash photolysis of the Ca2+ chelator diazo-2 increased by nearly twofold after D47A exchange. We conclude that the incorporation of the nondivalent cation-binding mutant of myosin RLC decreases the proportion of cycling cross-bridges in a force-generating state by decreasing the rate of formation of force-generating bridges and increasing the rate of detachment. These results suggest that divalent cation binding to myosin RLC plays an important role in modulating the kinetics of cross-bridge attachment and detachment.     

Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function

The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0 nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5 nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, “free” and “blocked”, formed an asymmetric structure named the “interacting-heads motif” (IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca²⁺-activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and post-tetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.     

History-dependent properties of skeletal muscle myofibrils contracting along the ascending limb of the force–length relationship

There is a history dependence of skeletal muscle contraction: stretching activated muscles induces a long-lasting force enhancement, while shortening activated muscles induces a long-lasting force depression. These history-dependent properties cannot be explained by the current model of muscle contraction, and its mechanism is unknown. The purposes of this study were (i) to evaluate if force enhancement and force depression are present at short lengths (the ascending limb of the force–length (FL) relationship), (ii) to evaluate if the history-dependent properties are associated with sarcomere length (SL) non-uniformity and (iii) to determine the effects of cross-bridge (de)activation on force depression. Rabbit psoas myofibrils were isolated and attached between two microneedles for force measurements. Images of the myofibrils were projected onto a linear photodiode array for measurements of SL. Myofibrils were activated by either Ca²⁺ or MgADP; the latter induces cross-bridge attachment to actin independently of Ca²⁺. Activated myofibrils were subjected to three stretches or shortenings (approx. 4% SL at approx. 0.07 µm s⁻¹ sarcomere⁻¹) along the ascending limb of the FL relationship separated by periods (approx. 5 s) of isometric contraction. Force after stretch was higher than force after shortening at similar SLs. The differences in force could not be explained by SL non-uniformity. The FL relationship produced by Ca²⁺- and MgADP-activated myofibrils were similar in stretch experiments, but after shortening MgADP activation produced forces that were higher than Ca²⁺ activation. Since MgADP induces the formation of strongly bound cross-bridges, this result suggests that force depression following shortening is associated with cross-bridge deactivation.     

Cooperative interactions between calcium-binding sites on glycerinated muscle fibers the influence of cross-bridge attachment

A double isotope technique and EGTA buffers were used to measure the binding of Ca²⁺ to rabbit psoas muscle fibers extracted with detergent and glycerol. These experiments were designed to test the effect of rigor complex formation, determined by the degree of filament overlap, on the properties of the Ca²⁺-binding sites in the intact filament lattice. In the presence of 5 mM MgCl₂ (no ATP), reduction of filament overlap was associated with a reduced binding of Ca²⁺ over the entire range of free Ca²⁺ concentrations (5 · 10^?8 – 2 · 10^?5 M). With maximum filament overlap (sarcomere length 2.1–2.2 μm) the maximum bound Ca²⁺ was equivalent to 4 mol Ca²⁺/mol troponin and there was significant positive interaction between binding sites, as shown by Scatchard and Hill plots. With no filament overlap (sarcomere length 3.8–4.4 μm) the maximum bound Ca²⁺ was equivalent to 3 μmol Ca²⁺/mol troponin and graphical analysis indicated a single class of non-interacting sites. The data provide evidence that when cross-bridge attachments between actin and myosin filaments are formed not only does an additional Ca²⁺ binding site appear, but cooperative properties are imposed upon the binding sites.     

Structural features of cross-bridges in isometrically contracting skeletal muscle

Two-dimensional x-ray diffraction was used to investigate structural features of cross-bridges that generate force in isometrically contracting skeletal muscle. Diffraction patterns were recorded from arrays of single, chemically skinned rabbit psoas muscle fibers during isometric force generation, under relaxation, and in rigor. In isometric contraction, a rather prominent intensification of the actin layer lines at 5.9 and 5.1 nm and of the first actin layer line at 37 nm was found compared with those under relaxing conditions. Surprisingly, during isometric contraction, the intensity profile of the 5.9-nm actin layer line was shifted toward the meridian, but the resulting intensity profile was different from that observed in rigor. We particularly addressed the question whether the differences seen between rigor and active contraction might be due to a rigor-like configuration of both myosin heads in the absence of nucleotide (rigor), whereas during active contraction only one head of each myosin molecule is in a rigor-like configuration and the second head is weakly bound. To investigate this question, we created different mixtures of weak binding myosin heads and rigor-like actomyosin complexes by titrating MgATPgammaS at saturating [Ca2+] into arrays of single muscle fibers. The resulting diffraction patterns were different in several respects from patterns recorded under isometric contraction, particularly in the intensity distribution along the 5.9-nm actin layer line. This result indicates that cross-bridges present during isometric force generation are not simply a mixture of weakly bound and single-headed rigor-like complexes but are rather distinctly different from the rigor-like cross-bridge. Experiments with myosin-S1 and truncated S1 (motor domain) support the idea that for a force generating cross-bridge, disorder due to elastic distortion might involve a larger part of the myosin head than for a nucleotide free, rigor cross-bridge.     

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.     

Effect of rigor and cycling cross-bridges on the structure of troponin C and on the Ca2+ affinity of the Ca2+-specific regulatory sites in skinned rabbit psoas fibers

Intrinsic troponin C (TnC) was extracted from small bundles of rabbit psoas fibers and replaced with TnC labeled with dansylaziridine (5-dimethylaminonaphthalene-1-sulfonyl). The flourescence of incorporated dansylaziridine-labeled TnC was enhanced by the binding of Ca2+ to the Ca2+-specific (regulatory) sites of TnC and was measured simultaneously with force (Zot, H.G., Güth, K., and Potter, J.D. (1986) J. Biol. Chem. 261, 15883-15890). Various myosin cross-bridge states also altered the fluorescence of dansylaziridine-labeled TnC in the filament, with cycling cross-bridges having a greater effect than rigor cross-bridges; and in both cases, there was an additional effect of Ca2+. The paired fluorescence and tension data were used to calculate the apparent Ca2+ affinity of the regulatory sites in the thin filament and were shown to increase at least 10-fold during muscle activation presumably due to the interaction of cycling cross-bridges with the thin filament. The cross-bridge state responsible for this enhanced Ca2+ affinity was shown to be the myosin-ADP state present only when cross-bridges are cycling. The steepness of the pCa force curves (where pCa represents the -log of the free Ca2+ concentration) obtained in the presence of ATP at short and long sarcomere lengths was the same, suggesting that cooperative interactions between adjacent troponin-tropomyosin units may spread along much of the actin filament when cross-bridges are attached to it. In contrast to the cycling cross-bridges, rigor bridges only increased the Ca2+ affinity of the regulatory sites 2-fold. Taken together, the results presented here indicate a strong coupling between the Ca2+ regulatory sites and cross-bridge interactions with the thin filament.     

Calmodulin and the regulation of smooth muscle contraction

Calmodulin, the ubiquitous and multifunctional Ca²⁺-binding protein, mediates many of the regulatory effects of Ca²⁺, including the contractile state of smooth muscle. The principal function of calmodulin in smooth muscle is to activate crossbridge cycling and the development of force in response to a [Ca²⁺]_i transientvia the activation of myosin light-chain kinase and phosphorylation of myosin. A distinct calmodulin-dependent kinase, Ca²⁺/calmodulin-dependent protein kinase II, has been implicated in modulation of smooth-muscle contraction. This kinase phosphorylates myosin light-chain kinase, resulting in an increase in the calmodulin concentration required for half-maximal activation of myosin light-chain kinase, and may account for desensitization of the contractile response to Ca²⁺. In addition, the thin filament-associated proteins, caldesmon and calponin, which inhibit the actin-activated MgATPase activity of smooth-muscle myosin (the cross-bridge cycling rate), appear to be regulated by calmodulin, either by the direct binding of Ca²⁺/calmodulin or indirectly by phosphorylation catalysed by Ca²⁺/calmodulin-dependent protein kinase II. Another level at which calmodulin can regulate smooth-muscle contraction involves proteins which control the movement of Ca²⁺ across the sarcolemmal and sarcoplasmic reticulum membranes and which are regulated by Ca²⁺/calmodulin, e.g. the sarcolemmal Ca²⁺ pump and the ryanodine receptor/Ca²⁺ release channel, and other proteins which indirectly regulate [Ca²⁺]_i via cyclic nucleotide synthesis and breakdown, e.g. NO synthase and cyclic nucleotide phosphodiesterase. The interplay of such regulatory mechanisms provides the flexibility and adaptability required for the normal functioning of smooth-muscle tissues.     

Nonlinear force-length relationship in the ADP-induced contraction of skeletal myofibrils

The regulatory mechanism of sarcomeric activity has not been fully clarified yet because of its complex and cooperative nature, which involves both Ca²⁺ and cross-bridge binding to the thin filament. To reveal the mechanism of regulation mediated by the cross-bridges, separately from the effect of Ca²⁺, we investigated the force-sarcomere length (SL) relationship in rabbit skeletal myofibrils (a single myofibril or a thin bundle) at SL > 2.2 μm in the absence of Ca²⁺ at various levels of activation by exogenous MgADP (4-20 mM) in the presence of 1 mM MgATP. The individual SLs were measured by phase-contrast microscopy to confirm the homogeneity of the striation pattern of sarcomeres during activation. We found that at partial activation with 4-8 mM MgADP, the developed force nonlinearly depended on the length of overlap between the thick and the thin filaments; that is, contrary to the maximal activation, the maximal active force was generated at shorter overlap. Besides, the active force became larger, whereas this nonlinearity tended to weaken, with either an increase in [MgADP] or the lateral osmotic compression of the myofilament lattice induced by the addition of a macromolecular compound, dextran T-500. The model analysis, which takes into account the [MgADP]-and the lattice-spacing-dependent probability of cross-bridge formation, was successfully applied to account for the force-SL relationship observed at partial activation. These results strongly suggest that the cross-bridge works as a cooperative activator, the function of which is highly sensitive to as little as ≤1 nm changes in the lattice spacing.     

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.     

Modulation of contractile activation in skeletal muscle by a calcium-insensitive troponin C mutant

Calcium controls the level of muscle activation via interactions with the troponin complex. Replacement of the native, skeletal calcium-binding subunit of troponin, troponin C, with mixtures of functional cardiac and mutant cardiac troponin C insensitive to calcium and permanently inactive provides a novel method to alter the number of myosin cross-bridges capable of binding to the actin filament. Extraction of skeletal troponin C and replacement with functional and mutant cardiac troponin C were used to evaluate the relationship between the extent of thin filament activation (fractional calcium binding), isometric force, and the rate of force generation in muscle fibers independent of the calcium concentration. The experiments showed a direct, linear relationship between force and the number of cross-bridges attaching to the thin filament. Further, above 35% maximal isometric activation, following partial replacement with mixtures of cardiac and mutant troponin C, the rate of force generation was independent of the number of actin sites available for cross-bridge interaction at saturating calcium concentrations. This contrasts with the marked decrease in the rate of force generation when force was reduced by decreasing the calcium concentration. The results are consistent with hypotheses proposing that calcium controls the transition between weakly and strongly bound cross-bridge states.