Regulation of force development studied by photolysis of caged ADP in rabbit skinned psoas fibers.

The present study examined the effects of Ca(2+) and strongly bound cross-bridges on tension development induced by changes in the concentration of MgADP. Addition of MgADP to the bath increased isometric tension over a wide range of [Ca(2+)] in skinned fibers from rabbit psoas muscle. Tension-pCa (pCa is -log [Ca(2+)]) relationships and stiffness measurements indicated that MgADP increased mean force per cross-bridge at maximal Ca(2+) and increased recruitment of cross-bridges at submaximal Ca(2+). Photolysis of caged ADP to cause a 0.5 mM MgADP jump initiated an increase in isometric tension under all conditions examined, even at pCa 6.4 where there was no active tension before ADP release. Tension increased monophasically with an observed rate constant, k(ADP), which was similar in rate and Ca(2+) sensitivity to the rate constant of tension re-development, k(tr), measured in the same fibers by a release-re-stretch protocol. The amplitude of the caged ADP tension transient had a bell-shaped dependence on Ca(2+), reaching a maximum at intermediate Ca(2+) (pCa 6). The role of strong binding cross-bridges in the ADP response was tested by treatment of fibers with a strong binding derivative of myosin subfragment 1 (NEM-S1). In the presence of NEM-S1, the rate and amplitude of the caged ADP response were no longer sensitive to variations in the level of activator Ca(2+). The results are consistent with a model in which ADP-bound cross-bridges cooperatively activate the thin filament regulatory system at submaximal Ca(2+). This cooperative interaction influences both the magnitude and kinetics of force generation in skeletal muscle.     

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.     

Acceleration of stretch activation in murine myocardium due to phosphorylation of myosin regulatory light chain

The regulatory light chains (RLCs) of vertebrate muscle myosins bind to the neck region of the heavy chain domain and are thought to play important structural roles in force transmission between the cross-bridge head and thick filament backbone. In vertebrate striated muscles, the RLCs are reversibly phosphorylated by a specific myosin light chain kinase (MLCK), and while phosphorylation has been shown to accelerate the kinetics of force development in skeletal muscle, the effects of RLC phosphorylation in cardiac muscle are not well understood. Here, we assessed the effects of RLC phosphorylation on force, and the kinetics of force development in myocardium was isolated in the presence of 2,3-butanedione monoxime (BDM) to dephosphorylate RLC, subsequently skinned, and then treated with MLCK to phosphorylate RLC. Since RLC phosphorylation may be an important determinant of stretch activation in myocardium, we recorded the force responses of skinned myocardium to sudden stretches of 1% of muscle length both before and after treatment with MLCK. MLCK increased RLC phosphorylation, increased the Ca(2+) sensitivity of isometric force, reduced the steepness of the force-pCa relationship, and increased both Ca(2+)-activated and Ca(2+)-independent force. Sudden stretch of myocardium during an otherwise isometric contraction resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed redevelopment of force, i.e., stretch activation, to levels greater than pre-stretch force. MLCK had profound effects on the stretch activation responses during maximal and submaximal activations: the amplitude and rate of force decay after stretch were significantly reduced, and the rate of delayed force recovery was accelerated and its amplitude reduced. These data show that RLC phosphorylation increases force and the rate of cross-bridge recruitment in murine myocardium, which would increase power generation in vivo and thereby enhance systolic function.     

Influence of a strong-binding myosin analogue on calcium-sensitive mechanical properties of skinned skeletal muscle fibers.

The ability of strong-binding myosin heads to activate the thin filament was investigated by incubating skinned single muscle fibers with N-ethylmaleimide-(NEM) modified myosin subfragment-1 (S1). Isometric force was influenced in a complex manner: during maximal calcium activation, NEM-S1 inhibited force with half-maximal inhibition at 20 microM while at submaximal calcium, NEM-S1 potentiated force with greatest effect at 6 microM. When fibers were treated with NEM-S1 (4-8 microM), the tension-pCa (-log [Ca2+]) relationship became less steep (i.e. the Hill coefficient decreased from 5.4 to 3.0 upon treatment with NEM-S1), but the midpoint was unchanged. These results support the idea that strong binding of intrinsic heads contributes to the cooperativity observed in Ca2+ activation of force. The NEM-S1-induced increase in force at low Ca2+ was associated with an acceleration of a kinetic transition, and this transition was activated to near maximum while force was not. The rate of force redevelopment following restretch (ktr) at submaximal calcium was increased by NEM-S1 in a concentration-dependent manner, yielding a maximum rate at low [Ca2+] which was similar to that observed during full activation. The effects of NEM-S1 on force and ktr indicate that strong-binding myosin cross-bridges are involved in activation of the thin filament.     

Ablation of myosin-binding protein-C accelerates force development in mouse myocardium

Myosin-binding protein-C (MyBP-C) is a thick filament-associated protein that binds tightly to myosin. Given that cMyBP-C may act to modulate cooperative activation of the thin filament by constraining the availability of myosin cross-bridges for binding to actin, we investigated the role of MyBP-C in the regulation of cardiac muscle contraction. We assessed the Ca(2+) sensitivity of force (pCa(50)) and the activation dependence of the rate of force redevelopment (k(tr)) in skinned myocardium isolated from wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)) mice. Mechanical measurements were performed at 22 degrees C in the absence and presence of a strong-binding, nonforce-generating analog of myosin subfragment-1 (NEM-S1). In the absence of NEM-S1, maximal force and k(tr) and the pCa(50) of isometric force did not differ between WT and cMyBP-C(-/-) myocardium; however, ablation of cMyBP-C-accelerated k(tr) at each submaximal force. Treatment of WT and cMyBP-C(-/-) myocardium with 3 muM NEM-S1 elicited similar increases in pCa(50,) but the effects of NEM-S1 to increase k(tr) at submaximal forces and thereby markedly reduce the activation dependence of k(tr) occurred to a greater degree in cMyBP-C(-/-) myocardium. Together, these results support the idea that cMyBP-C normally acts to constrain the interaction between myosin and actin, which in turn limits steady-state force development and the kinetics of cross-bridge interaction.     

Calcium-independent activation of skinned cardiac muscle by secophalloidin

Thin filament regulation of muscle contraction is believed to be mediated by both Ca2+ and strongly bound myosin cross-bridges. We found that secophalloidin (SPH, 5-8 mM) activates cross-bridge cycling without Ca2+ causing isometric force comparable to that induced by Ca2+. At saturated [SPH], Ca2+ further increased force by 20%. SPH-induced force was reversible upon washing with a relaxing solution. However, there was more than 30% irreversible loss in subsequent Ca2+-activated force. We hypothesize that SPH activates muscle via strongly bound cross-bridges. SPH-activated contraction provides a new model for studying the role of Ca2+ and cross-bridges in muscle regulation.     

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.     

Ca-activation and stretch-activation in insect flight muscle

Asynchronous insect flight muscle is specialized for myogenic oscillatory work, but can also produce isometric tetanic contraction. In skinned insect flight muscle fibers from Lethocerus, with sarcomere length monitored by a striation follower, we determined the relation between isometric force (F(0)) at serial increments of [Ca(2+)] and the additional active force recruited at each [Ca(2+)] by a stretch of approximately 12 nm per half-sarcomere (F(SA)). The isometric force-pCa relation shows that 1.5-2 units of pCa are necessary to raise isometric force from its threshold (pCa approximately 6.5) to its maximum (F(0,max)). The amplitude of F(SA) depends only on the preceding baseline level of isometric force, which must reach at least 0.05 F(0,max) to enable stretch-activation. F(SA) rises very steeply to its maximum as F(0) reaches approximately 0.2 F(0,max), then decreases as F(0) increases so as to produce a constant sum (F(0) + F(SA)) = F(max). Thus Ca- and stretch-activation are complementary pathways that trigger a common process of cross-bridge attachment and force production. We suggest that stretch-induced distortion of attached cross-bridges relieves the steric blocking by tropomyosin of additional binding sites on actin, thereby enabling maximum force even at low [Ca(2+)].     

Contributions of stretch activation to length-dependent contraction in murine myocardium

The steep relationship between systolic force production and end diastolic volume (Frank-Starling relationship) in myocardium is a potentially important mechanism by which the work capacity of the heart varies on a beat-to-beat basis, but the molecular basis for the effects of myocardial fiber length on cardiac work are still not well understood. Recent studies have suggested that an intrinsic property of myocardium, stretch activation, contributes to force generation during systolic ejection in myocardium. To examine the role of stretch activation in length dependence of activation we recorded the force responses of murine skinned myocardium to sudden stretches of 1% of muscle length at both short (1.90 microm) and long (2.25 microm) sarcomere lengths (SL). Maximal Ca(2+)-activated force and Ca(2+) sensitivity of force were greater at longer SL, such that more force was produced at a given Ca(2+) concentration. Sudden stretch of myocardium during an otherwise isometric contraction resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed development of force, i.e., stretch activation, to levels greater than prestretch force. At both maximal and submaximal activations, increased SL significantly reduced the initial rate of force decay following stretch; at submaximal activations (but not at maximal) the rate of delayed force development was accelerated. This combination of mechanical effects of increased SL would be expected to increase force generation during systolic ejection in vivo and prolong the period of ejection. These results suggest that sarcomere length dependence of stretch activation contributes to the steepness of the Frank-Starling relationship in living myocardium.     

The effects of force inhibition by sodium vanadate on cross-bridge binding, force redevelopment, and Ca2+ activation in cardiac muscle

Strongly bound, force-generating myosin cross-bridges play an important role as allosteric activators of cardiac thin filaments. Sodium vanadate (Vi) is a phosphate analog that inhibits force by preventing cross-bridge transition into force-producing states. This study characterizes the mechanical state of cross-bridges with bound Vi as a tool to examine the contribution of cross-bridges to cardiac contractile activation. The K(i) of force inhibition by Vi was approximately 40 microM. Sinusoidal stiffness was inhibited with Vi, although to a lesser extent than force. We used chord stiffness measurements to monitor Vi-induced changes in cross-bridge attachment/detachment kinetics at saturating [Ca(2+)]. Vi decreased chord stiffness at the fastest rates of stretch, whereas at slow rates chord stiffness actually increased. This suggests a shift in cross-bridge population toward low force states with very slow attachment/detachment kinetics. Low angle x-ray diffraction measurements indicate that with Vi cross-bridge mass shifted away from thin filaments, implying decreased cross-bridge/thin filament interaction. The combined x-ray and mechanical data suggest at least two cross-bridge populations with Vi; one characteristic of normal cycling cross-bridges, and a population of weak-binding cross-bridges with bound Vi and slow attachment/detachment kinetics. The Ca(2+) sensitivity of force (pCa(50)) and force redevelopment kinetics (k(TR)) were measured to study the effects of Vi on contractile activation. When maximal force was inhibited by 40% with Vi pCa(50) decreased, but greater force inhibition at higher [Vi] did not further alter pCa(50). In contrast, the Ca(2+) sensitivity of k(TR) was unaffected by Vi. Interestingly, when force was inhibited by Vi k(TR) increased at submaximal levels of Ca(2+)-activated force. Additionally, k(TR) is faster at saturating Ca(2+) at [Vi] that inhibit force by > approximately 70%. The effects of Vi on k(TR) imply that k(TR) is determined not only by the intrinsic properties of the cross-bridge cycle, but also by cross-bridge contribution to thin filament activation.     

Force enhancement and relaxation rates after stretch of activated muscle fibres

The residual force enhancement following muscle stretch might be associated with an increase in the proportion of attached cross-bridges, as supported by stiffness measurements. In this case, it could be caused by an increase in the attachment or a decrease in the detachment rate of cross-bridges, or a combination of the two. The purpose of this study was to investigate if the stretch-induced force enhancement is related to cross-bridge attachment/detachment kinetics. Single muscle fibres dissected from the lumbrical muscle of frog were place at a length approximately 20% longer than the plateau of the force-length relationship; they were maximally activated, and after full isometric force was reached, ramp stretches were imposed with amplitudes of 5 and 10% fibre length, at a speed of 40% fibre length s(-1). Experiments were performed in Ringer's solution, and with the addition of 2, 5 and 10 nM of 2,3-butanedione monoxime (BDM), a drug that places cross-bridges in a pre-power-stroke, state, inhibiting force production. The total force following stretch was higher than the corresponding force measured after isometric contraction at the corresponding length. This residual force enhancement was accompanied by an increase relaxation time. BDM, which decreases force production during isometric contractions, considerably increased the relative levels of force enhancement. BDM also increased relaxation times after stretch, beyond the levels observed during reference contractions in Ringer's solution, and beyond isometric control tests at the corresponding BDM concentrations. Together, these results support the idea that force enhancement is caused, at least in part, by a decrease in cross-bridge detachment rates, as manifested by the increased relaxation times following fibre stretch.     

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.     

Cross-bridge mechanism of residual force enhancement after stretching in a skeletal muscle

A muscle model that uses a modified Langevin equation with actomyosin potentials was used to describe the residual force enhancement after active stretching. Considering that the new model uses cross-bridge theory to describe the residual force enhancement, it is different from other models that use passive stretching elements. Residual force enhancement was simulated using a half sarcomere comprising 100 myosin molecules. In this paper, impulse is defined as the integral of an excess force from the steady isometric force over the time interval for which a stretch is applied. The impulse was calculated from the force response due to fast and slow muscle stretches to demonstrate the viscoelastic property of the cross-bridges. A cross-bridge mechanism was proposed as a way to describe the residual force enhancement on the basis of the impulse results with reference to the compliance of the actin filament. It was assumed that the period of the actin potential increased by 0.5% and the amplitude of the potential decreased by 0.5% when the half sarcomere was stretched by 10%. The residual force enhancement after 21.0% sarcomere stretching was 6.9% of the maximum isometric force of the muscle; this value was due to the increase in the number of cross-bridges.     

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.     

Mechanical transients initiated by photolysis of caged ATP within fibers of insect fibrillar flight muscle

Kinetics of the cross-bridge cycle in insect fibrillar flight muscle have been measured using laser pulse photolysis of caged ATP and caged inorganic phosphate (Pi) to produce rapid step increases in the concentration of ATP and Pi within single glycerol-extracted fibers. Rapid photochemical liberation of 100 microM-1 mM ATP from caged ATP within a fiber caused relaxation in the absence of Ca2+ and initiated an active contraction in the presence of approximately 30 microM Ca2+. The apparent second order rate constant for detachment of rigor cross-bridges by ATP was between 5 x 10(4) and 2 x 10(5) M-1s-1. This rate is not appreciably sensitive to the Ca2+ or Pi concentrations or to rigor tension level. The value is within an order of magnitude of the analogous reaction rate constant measured with isolated actin and insect myosin subfragment-1 (1986. J. Muscle Res. Cell Motil. 7:179-192). In both the absence and presence of Ca2+ insect fibers showed evidence of transient cross-bridge reattachment after ATP-induced detachment from rigor, as found in corresponding experiments on rabbit psoas fibers. However, in contrast to results with rabbit fibers, tension traces of insect fibers starting at different rigor tensions did not converge to a common time course until late in the transients. This result suggests that the proportion of myosin cross-bridges that can reattach into force-generating states depends on stress or strain in the filament lattice. A steady 10-mM concentration of Pi markedly decreased the transient reattachment phase after caged ATP photolysis. Pi also decreased the amplitude of stretch activation after step stretches applied in the presence of Ca2+ and ATP. Photolysis of caged Pi during stretch activation abruptly terminated the development of tension. These results are consistent with a linkage between Pi release and the steps leading to force production in the cross-bridge cycle.     

Stretch of active muscle during the declining phase of the calcium transient produces biphasic changes in calcium binding to the activating sites

In voltage-clamped barnacle single muscle fibers, muscle shortening during the declining phase of the calcium transient increases myoplasmic calcium. This extra calcium is probably released from the activating sites by a change in affinity when cross-bridges break (Gordon, A. M., and E. B. Ridgway, 1987. J. Gen. Physiol. 90:321-340). Stretching the muscle at similar times causes a more complex response, a rapid increase in intracellular calcium followed by a transient decrease. The amplitudes of both phases increase with the rate and amplitude of stretch. The rapid increase, however, appears only when the muscle is stretched more than approximately 0.4%. This is above the length change that produces the breakpoint in the force record during a ramp stretch. This positive phase in response to large stretches is similar to that seen on equivalent shortening at the same point in the contraction. For stretches at different times during the calcium transient, the peak amplitude of the positive phase has a time course that is delayed relative to the calcium transient, while the peak decrease during the negative phase has an earlier time course that is more similar to the calcium transient. The amplitudes of both phases increase with increasing strength of stimulation and consequent force. When the initial muscle the active force. A large decrease in length (which drops the active force to zero) decreases the extra calcium seen on a subsequent restretch. After such a shortening step, the extra calcium on stretch recovers (50 ms half time) toward the control level with the same time course as the redeveloped force. Conversely, stretching an active fiber decreases the extra calcium on a subsequent shortening step that is imposed shortly afterward. Enhanced calcium binding due to increased length alone cannot explain our data. We hypothesize that the calcium affinity of the activating sites increases with cross-bridge attachment and further with cross-bridge strain. This accounts for the biphasic response to stretch as follows: cross-bridges detached by stretch first decrease calcium affinity, then upon reattachment increase calcium affinity due to the strained configuration brought on by the stretch. The experiments suggest that cross-bridge attachment and strain can modify calcium binding to the activating sites in intact muscle.     

Rate constant of muscle force redevelopment reflects cooperative activation as well as cross-bridge kinetics.

The rate of muscle force redevelopment after release-restretch protocols has previously been interpreted using a simple two-state cross-bridge cycling model with rate constants for transitions between non-force-bearing and force-bearing states, f, and between force-bearing and non-force-bearing states, g. Changes in the rate constant of force redevelopment, as with varying levels of Ca2+ activation, have traditionally been attributed to Ca(2+)-dependent f. The current work adds to this original model a state of unactivated, noncycling cross-bridges. The resulting differential equation for activated, force-bearing cross-bridges, Ncf, was Ncf = -[g+f(K/(K + 1))] Ncf+f(K/(K + 1))NT, where K is an equilibrium constant defining the distribution between cycling and noncycling cross-bridges and NT is the total number of cross-bridges. Cooperativity by which force-bearing cross-bridges participate in their own activation was introduced by making K depend on Ncf. Model results demonstrated that such cooperativity, which tends to enhance force generation at low levels of Ca2+ activation, has a counter-intuitive effect of slowing force redevelopment. These dynamic effects of cooperativity are most pronounced at low Ca2+ activation. As Ca2+ activation increases, the cooperative effects become less important to the dynamics of force redevelopment and, at the highest levels of Ca2+ activation, the dynamics of force redevelopment reflect factors other than cooperative mechanisms. These results expand on earlier interpretations of Ca2+ dependence of force redevelopment; rather than Ca(2+)-dependent f, Ca(2+)-dependent force redevelopment arises from changing expressions of cooperativity between force-bearing cross-bridges and activation.     

Expression of cardiac troponin T with COOH-terminal truncation accelerates cross-bridge interaction kinetics in mouse myocardium

Transgenic mice expressing an allele of cardiac troponin T (cTnT) with a COOH-terminal truncation (cTnT(trunc)) exhibit severe diastolic and mild systolic dysfunction. We tested the hypothesis that contractile dysfunction in myocardium expressing low levels of cTnT(trunc) (i.e., <5%) is due to slowed cross-bridge kinetics and reduced thin filament activation as a consequence of reduced cross-bridge binding. We measured the Ca(2+) sensitivity of force development [pCa for half-maximal tension generation (pCa(50))] and the rate constant of force redevelopment (k(tr)) in cTnT(trunc) and wild-type (WT) skinned myocardium both in the absence and in the presence of a strong-binding, non-force-generating derivative of myosin subfragment-1 (NEM-S1). Compared with WT mice, cTnT(trunc) mice exhibited greater pCa(50), reduced steepness of the force-pCa relationship [Hill coefficient (n(H))], and faster k(tr) at submaximal Ca(2+) concentration ([Ca(2+)]), i.e., reduced activation dependence of k(tr). Treatment with NEM-S1 elicited similar increases in pCa(50) and similar reductions in n(H) in WT and cTnT(trunc) myocardium but elicited greater increases in k(tr) at submaximal activation in cTnT(trunc) myocardium. Contrary to our initial hypothesis, cTnT(trunc) appears to enhance thin filament activation in myocardium, which is manifested as significant increases in Ca(2+)-activated force and the rate of cross-bridge attachment at submaximal [Ca(2+)]. Although these mechanisms would not be expected to depress systolic function per se in cTnT(trunc) hearts, they would account for slowed rates of myocardial relaxation during early diastole.     

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

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

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

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