Cooperative Activation Of Thin Filament Research Articles

Effects of omecamtiv mecarbil on the contractile properties of skinned porcine left atrial and ventricular muscles.

Omecamtiv mecarbil (OM) is a novel inotropic agent for heart failure with systolic dysfunction. OM prolongs the actomyosin attachment duration, which enhances thin filament cooperative activation and accordingly promotes the binding of neighboring myosin to actin. In the present study, we investigated the effects of OM on the steady-state contractile properties in skinned porcine left ventricular (PLV) and atrial (PLA) muscles. OM increased Ca2+ sensitivity in a concentration-dependent manner in PLV, by left shifting the mid-point (pCa50) of the force-pCa curve (ΔpCa50) by ∼0.16 and ∼0.33 pCa units at 0.5 and 1.0 μM, respectively. The Ca2+-sensitizing effect was likewise observed in PLA, but less pronounced with ΔpCa50 values of ∼0.08 and ∼0.22 pCa units at 0.5 and 1.0 μM, respectively. The Ca2+-sensitizing effect of OM (1.0 μM) was attenuated under enhanced thin filament cooperative activation in both PLV and PLA; this attenuation occurred directly via treatment with fast skeletal troponin (ΔpCa50: ∼0.16 and ∼0.10 pCa units in PLV and PLA, respectively) and indirectly by increasing the number of strongly bound cross-bridges in the presence of 3 mM MgADP (ΔpCa50: ∼0.21 and ∼0.08 pCa units in PLV and PLA, respectively). It is likely that this attenuation of the Ca2+-sensitizing effect of OM is due to a decrease in the number of “recruitable” cross-bridges that can potentially produce active force. When cross-bridge detachment was accelerated in the presence of 20 mM inorganic phosphate, the Ca2+-sensitizing effect of OM (1.0 μM) was markedly decreased in both types of preparations (ΔpCa50: ∼0.09 and ∼0.03 pCa units in PLV and PLA, respectively). The present findings suggest that the positive inotropy of OM is more markedly exerted in the ventricle than in the atrium, which results from the strongly bound cross-bridge-dependent allosteric activation of thin filaments.

Positive cardiac inotrope omecamtiv mecarbil activates muscle despite suppressing the myosin working stroke

Omecamtiv mecarbil (OM) is a positive cardiac inotrope in phase-3 clinical trials for treatment of heart failure. Although initially described as a direct myosin activator, subsequent studies are at odds with this description and do not explain OM-mediated increases in cardiac performance. Here we show, via single-molecule, biophysical experiments on cardiac myosin, that OM suppresses myosin’s working stroke and prolongs actomyosin attachment 5-fold, which explains inhibitory actions of the drug observed in vitro. OM also causes the actin-detachment rate to become independent of both applied load and ATP concentration. Surprisingly, increased myocardial force output in the presence of OM can be explained by cooperative thin-filament activation by OM-inhibited myosin molecules. Selective suppression of myosin is an unanticipated route to muscle activation that may guide future development of therapeutic drugs.

Molecule specific effects of PKA-mediated phosphorylation on rat isolated heart and cardiac myofibrillar function

Increased cardiac myocyte contractility by the β-adrenergic system is an important mechanism to elevate cardiac output to meet hemodynamic demands and this process is depressed in failing hearts. While increased contractility involves augmented myoplasmic calcium transients, the myofilaments also adapt to boost the transduction of the calcium signal. Accordingly, ventricular contractility was found to be tightly correlated with PKA-mediated phosphorylation of two myofibrillar proteins, cardiac myosin binding protein-C (cMyBP-C) and cardiac troponin I (cTnI), implicating these two proteins as important transducers of hemodynamics to the cardiac sarcomere. Consistent with this, we have previously found that phosphorylation of myofilament proteins by PKA (a downstream signaling molecule of the beta-adrenergic system) increased force, slowed force development rates, sped loaded shortening, and increased power output in rat skinned cardiac myocyte preparations. Here, we sought to define molecule-specific mechanisms by which PKA-mediated phosphorylation regulates these contractile properties. Regarding cTnI, the incorporation of thin filaments with unphosphorylated cTnI decreased isometric force production and these changes were reversed by PKA-mediated phosphorylation in skinned cardiac myocytes. Further, incorporation of unphosphorylated cTnI sped rates of force development, which suggests less cooperative thin filament activation and reduced recruitment of non-cycling cross-bridges into the pool of cycling cross-bridges, a process that would tend to depress both myocyte force and power. Regarding MyBP-C, PKA treatment of slow-twitch skeletal muscle fibers caused phosphorylation of MyBP-C (but not slow skeletal TnI (ssTnI)) and yielded faster loaded shortening velocity and ∼30% increase in power output. These results add novel insight into the molecular specificity by which the β-adrenergic system regulates myofibrillar contractility and how attenuation of PKA-induced phosphorylation of cMyBP-C and cTnI may contribute to ventricular pump failure.

Molecule Specific Effects of PKA-Mediated Phosphorylation on Myofibrillar Function

Increased cardiac myocyte contractility by the beta-adrenergic system is an important mechanism to elevate cardiac output to meet greater hemodynamic load and this process is often depressed in failing hearts. While increased contractility involves augmented myoplasmic calcium transients, the myofilaments also adapt to boost the transduction of the calcium signal. Accordingly, ventricular contractility is tightly correlated with PKA-mediated phosphorylation of two myofibrillar proteins, cardiac myosin binding protein-C (cMyBP-C) and cardiac troponin I (cTnI), implicating these two proteins as important transducers of hemodynamics to the cardiac sarcomere. Consistent with this, we have previously found that phosphorylation of myofilament proteins by PKA (a downstream signaling molecule of the beta-adrenergic system) increased force, slowed force development rates, sped loaded shortening, and increased power output in rat skinned cardiac myocyte preparations. Here, we sought to define molecule-specific mechanisms by which PKA-mediated phosphorylation modulates these contractile properties. Regarding cTnI, the incorporation of thin filaments with a majority of unphosphorylated cTnI (as observed in some models of late stage heart failure) decreased isometric force production at any given activator [Ca2+] and these changes were reversed by PKA-mediated phosphorylation in skinned cardiac myocytes. In addition, incorporation of unphosphorylated cTnI sped rates of force development, which suggests less cooperative thin filament activation and recruitment of non-cycling cross-bridges into the pool of cycling cross-bridges, a process that would tend to depress myocyte force and power. Regarding cMyBP-C, PKA treatment of slow-twitch skeletal muscle fibers caused phosphorylation of MyBP-C (but not TnI) and yielded faster loaded shortening velocity and an ∼30% increase in power output. These results add novel insight into the molecular specificity by which the beta-adrenergic system controls myofibrillar contractility and how attenuation of PKA-induced phosphorylation of cMyBP-C and cTnI may contribute to ventricular pump failure.

Depressed Frank–Starling mechanism in the left ventricular muscle of the knock-in mouse model of dilated cardiomyopathy with troponin T deletion mutation ΔK210

It has been reported that the Frank–Starling mechanism is coordinately regulated in cardiac muscle via thin filament “on–off” equilibrium and titin-based lattice spacing changes. In the present study, we tested the hypothesis that the deletion mutation ΔK210 in the cardiac troponin T gene shifts the equilibrium toward the “off” state and accordingly attenuate the sarcomere length (SL) dependence of active force production, via reduced cross-bridge formation. Confocal imaging in isolated hearts revealed that the cardiomyocytes were enlarged, especially in the longitudinal direction, in ΔK210 hearts, with striation patterns similar to those in wild type (WT) hearts, suggesting that the number of sarcomeres is increased in cardiomyocytes but the sarcomere length remains unaltered. For analysis of the SL dependence of active force, skinned muscle preparations were obtained from the left ventricle of WT and knock-in (ΔK210) mice. An increase in SL from 1.90 to 2.20μm shifted the mid-point (pCa50) of the force-pCa curve leftward by ~0.21pCa units in WT preparations. In ΔK210 muscles, Ca2+ sensitivity was lower by ~0.37pCa units, and the SL-dependent shift of pCa50, i.e., ΔpCa50, was less pronounced (~0.11pCa units), with and without protein kinase A treatment. The rate of active force redevelopment was lower in ΔK210 preparations than in WT preparations, showing blunted thin filament cooperative activation. An increase in thin filament cooperative activation upon an increase in the fraction of strongly bound cross-bridges by MgADP increased ΔpCa50 to ~0.21pCa units. The depressed Frank–Starling mechanism in ΔK210 hearts is the result of a reduction in thin filament cooperative activation.

Congenital myopathy-causing tropomyosin mutations induce thin filament dysfunction via distinct physiological mechanisms

In humans, congenital myopathy-linked tropomyosin mutations lead to skeletal muscle dysfunction, but the cellular and molecular mechanisms underlying such dysfunction remain obscure. Recent studies have suggested a unifying mechanism by which tropomyosin mutations partially inhibit thin filament activation and prevent proper formation and cycling of myosin cross-bridges, inducing force deficits at the fiber and whole-muscle levels. Here, we aimed to verify this mechanism using single membrane-permeabilized fibers from patients with three tropomyosin mutations (TPM2-null, TPM3-R167H and TPM2-E181K) and measuring a broad range of parameters. Interestingly, we identified two divergent, mutation-specific pathophysiological mechanisms. (i) The TPM2-null and TPM3-R167H mutations both decreased cooperative thin filament activation in combination with reductions in the myosin cross-bridge number and force production. The TPM3-R167H mutation also induced a concomitant reduction in thin filament length. (ii) In contrast, the TPM2-E181K mutation increased thin filament activation, cross-bridge binding and force generation. In the former mechanism, modulating thin filament activation by administering troponin activators (CK-1909178 and EMD 57033) to single membrane-permeabilized fibers carrying tropomyosin mutations rescued the thin filament activation defect associated with the pathophysiology. Therefore, administration of troponin activators may constitute a promising therapeutic approach in the future.

Sarcomere length-dependent Ca2+ activation in skinned rabbit psoas muscle fibers: coordinated regulation of thin filament cooperative activation and passive force

In skeletal muscle, active force production varies as a function of sarcomere length (SL). It has been considered that this SL dependence results simply from a change in the overlap length between the thick and thin filaments. The purpose of this study was to provide a systematic understanding of the SL-dependent increase in Ca2+ sensitivity in skeletal muscle, by investigating how thin filament “on–off” switching and passive force are involved in the regulation. Rabbit psoas muscles were skinned, and active force measurements were taken at various Ca2+ concentrations with single fibers, in the short (2.0 and 2.4 μm) and long (2.4 and 2.8 μm) SL ranges. Despite the same magnitude of SL elongation, the SL-dependent increase in Ca2+ sensitivity was more pronounced in the long SL range. MgADP (3 mM) increased the rate of rise of active force and attenuated SL-dependent Ca2+ activation in both SL ranges. Conversely, inorganic phosphate (Pi, 20 mM) decreased the rate of rise of active force and enhanced SL-dependent Ca2+ activation in both SL ranges. Our analyses revealed that, in the absence and presence of MgADP or Pi, the magnitude of SL-dependent Ca2+ activation was (1) inversely correlated with the rate of rise of active force, and (2) in proportion to passive force. These findings suggest that the SL dependence of active force in skeletal muscle is regulated via thin filament “on–off” switching and titin (connectin)-based interfilament lattice spacing modulation in a coordinated fashion, in addition to the regulation via the filament overlap.Electronic supplementary materialThe online version of this article (doi:10.1007/s12576-011-0173-8) contains supplementary material, which is available to authorized users.

Reducing Thin Filament Ca 2+ Affinity with a cTnC Variant (L57Q) Reduces Force but Enhances Cross-Bridge Dependence of Cooperative Activation in Demembranated Rat Trabeculae

Activation of cardiac contraction is initiated by binding of Ca2+ to troponin C (cTnC) and regulated by cooperative strong cross-bridge binding. We previously showed that passive exchange with cTn containing a cTnC variant, L48Q, increased Ca2+ sensitivity of force development and eliminated sarcomere length (SL) dependence of Ca2+ sensitivity of force in rat cardiac trabeculae. This was shown to be due to decreased reliance on strong cross-bridge binding for full thin-filament activation, possibly due to stronger cTnC-cTnI interaction. We also showed that PKA phosphorylation decreased Ca2+ sensitivity of force and eliminated SL dependence of force-pCa relations, by unknown specific mechanisms. Here we test the hypothesis that incorporation of a cTnC variant with decreased Ca2+ binding affinity, L57Q cTnC, will result in increased reliance on cross-bridge binding for full activation, thus increasing SL-dependence of Ca2+ sensitivity. As expected, results indicate trabeculae passively exchanged with L57Q cTnC-cTn displayed decreased Ca2+ sensitivity and rate of force production compared to WT cTnC at a given [Ca2+]. Interestingly, preliminary results indicate L57Q cTnC-cTn mildly increased the SL-dependence of Ca2+ sensitivity of force and also significantly decreased maximal force. Both of these effects were not observed in PKA-treated trabeculae, which had a comparable reduction in Ca2+ sensitivity of force. These findings suggest that reducing cTnC Ca2+ affinity per se can reduce Ca2+ sensitivity of contractile activation to the point of limited overall force production, which may enhance cross-bridge dependence of cooperative thin filament activation. Current experiments aim to increase force in L57Q cTnC (to near WT cTnC) by increasing cross-bridge attachment using 2’-deoxy-ATP, which has previously been shown to increase force and Ca2+ sensitivity while maintaining SL dependence. NIH-HL65497(MR), AHA-2310117(FSK).

Regulatory mechanism of length-dependent activation in skinned porcine ventricular muscle: role of thin filament cooperative activation in the Frank-Starling relation

Cardiac sarcomeres produce greater active force in response to stretch, forming the basis of the Frank-Starling mechanism of the heart. The purpose of this study was to provide the systematic understanding of length-dependent activation by investigating experimentally and mathematically how the thin filament “on–off” switching mechanism is involved in its regulation. Porcine left ventricular muscles were skinned, and force measurements were performed at short (1.9 µm) and long (2.3 µm) sarcomere lengths. We found that 3 mM MgADP increased Ca2+ sensitivity of force and the rate of rise of active force, consistent with the increase in thin filament cooperative activation. MgADP attenuated length-dependent activation with and without thin filament reconstitution with the fast skeletal troponin complex (sTn). Conversely, 20 mM of inorganic phosphate (Pi) decreased Ca2+ sensitivity of force and the rate of rise of active force, consistent with the decrease in thin filament cooperative activation. Pi enhanced length-dependent activation with and without sTn reconstitution. Linear regression analysis revealed that the magnitude of length-dependent activation was inversely correlated with the rate of rise of active force. These results were quantitatively simulated by a model that incorporates the Ca2+-dependent on–off switching of the thin filament state and interfilament lattice spacing modulation. Our model analysis revealed that the cooperativity of the thin filament on–off switching, but not the Ca2+-binding ability, determines the magnitude of the Frank-Starling effect. These findings demonstrate that the Frank-Starling relation is strongly influenced by thin filament cooperative activation.

The Combined Effects of ADP, ATP, and Myosin Density on Cooperative Activation of Thin Filaments

Cooperative activation of thin filaments has been achieved by decreasing [ATP], increasing [ADP], and increasing myosin densities; however, these observations have yet to be incorporated into a self-consistent description of thin filament regulation. It has been noted in previous skinned muscle fiber studies that contraction can be initiated with high [ADP] when Ca2+ is absent. Similarly, low [ATP] has been shown to activate thin filaments in Ca2+-free motility assays. Based on these observations, we hypothesize that high concentrations of ADP can cause thin filament cooperative activation in the absence of Ca2+. Using an in vitro motility assay, we determine the effects of ATP, ADP, and myosin density on the cooperative activation of myosin-based thin filament sliding, V. At 1mM [ATP], V is activated over an [ADP] range from ∼2 to 8mM and becomes increasingly inhibited at [ADP] >8mM. This biphasic effect of [ADP] on V is similar to the effects of [ATP] on cooperative thin filament activation previously measured in vitro. The observed effects of [ADP] and [ATP] on V are consistent with a model in which increasing [ADP] or decreasing [ATP] increase the probability that a myosin head is bound within a thin filament regulatory unit, cooperatively activating that regulatory unit (and possible adjacent ones) and increasing V. At sufficiently high [ADP] or sufficiently low [ATP], the thin filament becomes fully activated, and a further increase in [ADP] or decrease in [ATP] slows V by inhibiting detachment kinetics.

Impact of Ischemia/Reperfusion Associated TnI Degradation on Cross-Bridge Dynamics in Skinned Rat Cardiac Trabeculae

Proteolytic degradation of Troponin I (cTnI) may be the cause for the depressed contractility that is seen in myocardial stunning. Here, we studied the impact of a proteolytic fragment cTnI63-193 (identified by McDonough et al, 1999) on cross-bridge cycling dynamics in isolated myocardium. Murine cTnI63-193 mutant, as well as wild type cTnC and cTnT, were expressed in E.coli. Next, FPLC purified Troponin (cTn) complex containing either wild type cTnI or cTnI63-193 was exchanged for endogenous cTn in skinned rat cardiac trabeculae; Western blot analysis confirmed that >75% cTn was exchanged. Myofilament chemo-mechanical cross-bridge dynamics were determined as function of [Ca2+] at SL=2.2 µm using an enzyme-coupled UV absorbance technique (de Tombe & Stienen, 1995).Compared to wild-type exchange, cTnI63-193 exchanged fibers displayed ∼30% decrease in myofilament Ca2+ sensitivity (EC50). In contrast, neither maximal tension development, nor maximal ATPase activity (and consequently tension-cost), were significant different between the groups, nor was cooperative thin filament activation (Hill coefficient). We conclude that cTnI63-193 found following severe ischemic/reperfusion affects cardiac function predominantly via decreased myofilament Ca2+-sensitivity. Our results may benefit rational drug development aimed to prevent ischemic/reperfusion injury in patients.

Mechanical and kinetic effects of shortened tropomyosin reconstituted into myofibrils

The effects of tropomyosin on muscle mechanics and kinetics were examined in skeletal myofibrils using a novel method to remove tropomyosin (Tm) and troponin (Tn) and then replace these proteins with altered versions. Extraction employed a low ionic strength rigor solution, followed by sequential reconstitution at physiological ionic strength with Tm then Tn. SDS-PAGE analysis was consistent with full reconstitution, and fluorescence imaging after reconstitution using Oregon-green-labeled Tm indicated the expected localization. Myofibrils remained mechanically viable: maximum isometric forces of myofibrils after sTm/sTn reconstitution (control) were comparable (~84%) to the forces generated by non-reconstituted preparations, and the reconstitution minimally affected the rate of isometric activation (kact), calcium sensitivity (pCa50), and cooperativity (nH). Reconstitutions using various combinations of cardiac and skeletal Tm and Tn indicated that isoforms of both Tm and Tn influence calcium sensitivity of force development in opposite directions, but the isoforms do not otherwise alter cross-bridge kinetics. Myofibrils reconstituted with Δ23Tm, a deletion mutant lacking the second and third of Tm’s seven quasi-repeats, exhibited greatly depressed maximal force, moderately slower kact rates and reduced nH. Δ23Tm similarly decreased the cooperativity of calcium binding to the troponin regulatory sites of isolated thin filaments in solution. The mechanisms behind these effects of Δ23Tm also were investigated using Pi and ADP jumps. Pi and ADP kinetics were indistinguishable in Δ23Tm myofibrils compared to controls. The results suggest that the deleted region of tropomyosin is important for cooperative thin filament activation by calcium.

Role of Thin Filament Cooperative Activation in Length-dependent Activation in Skinned Porcine Ventricular Muscle

The basis of the Frank-Starling mechanism of the heart is the intrinsic ability of cardiac sarcomeres to produce greater active force in response to stretch (i.e., length-dependent activation). We have reported that troponin plays a key role in this phenomenon via on-off switching of the thin filament state, in concert with titin-based passive force (Terui et al., J Gen. Physiol. 131;275-283:2008). In the present study, we systematically investigated the role of thin filament cooperative activation in length-dependent activation using skinned porcine ventricular muscle at sarcomere lengths of 1.9 and 2.3 μm. MgADP (3 mM) increased Ca2+ sensitivity of force and enhanced the speed of contraction, indicating enhanced cooperative activation. MgADP was found to attenuate length-dependent activation, with and without quasi-complete reconstitution of thin filaments with fast skeletal troponin (sTn; from rabbits). Conversely, inorganic phosphate (Pi, 20 mM) decreased Ca2+ sensitivity of force and the speed of contraction, indicating reduced cooperative activation. Pi enhanced length-dependent activation, with and without sTn reconstitution. Qualitatively similar results were obtained with MgADP or Pi in rabbit fast skeletal muscle, with higher Ca2+ sensitivity of force than in cardiac muscle. Linear regression analysis revealed that the speed of contraction, Ca2+ sensitivity of force and length-dependent activation were strongly correlated in both cardiac and skeletal muscle. These results suggest that length-dependent activation is regulated via thin filament cooperative activation, such that the length-dependent increase in the fraction of cross-bridges is less in high cooperative activation states, coupled with a loss of recruitable cross-bridges.

Sarcomere Length Dependence Of The Force-pCa Relationship In Cardiac Muscle Is Influenced By Properties Of Thin Filament Regulatory Units

Myocardial performance is tightly regulated by sarcomere length (SL), which is thought to be due at least in part to concurrent changes in myofilament lattice spacing and the relative proximity (and binding probability) of myosin heads to actin. Furthermore, crossbridge binding enhances Ca2+ binding to troponin (Tn) in cardiac muscle, a unique form of cooperative thin filament activation whose mechanism is currently unknown. Experiments here were designed to test the hypothesis that the properties of Tn are important in determining the SL dependence of force in cardiac muscle. We compared trabeculae exchanged with WT cTn vs. cTn containing a mutant (L48Q) cTnC with enhanced Ca2+ affinity. L48Q cTnC caused a left-shift in the force-pCa relationship at long (2.3 μm) SL as compared to WT cTnC. Interestingly, L48Q cTnC effectively eliminated SL-dependence of the force-pCa relationship, via a much larger left-shift at short SL, while SL dependence of Fmax was unaffected. This suggests that SL-dependence of cardiac force development can be greatly influenced by the properties of native cTn, perhaps by limiting crossbridge binding, and that this effect is likely most important at shorter SL. Furthermore, increasing the Ca2+ binding and/or cTnC-cTnI interaction properties of cTn (such as with L48Q cTnC) can reduce or eliminate this limitation. Ongoing experiments will determine whether TnI phosphorylation can restore SL dependence by decreasing Tn Ca2+ affinity, thereby reducing myosin access to actin binding sites. This will also be tested by exchange with I61Q cTnC-Tn, a mutant with reduced Ca2+ affinity. Overall, these results imply that the cardiac length-force relationship is governed, at least in part, by properties of thin filament regulatory units. Support appreciated from NIH R01 HL 65497 (MR) and T32 HL07828 (FSK).

Influence of enhanced troponin C Ca2+‐binding affinity on cooperative thin filament activation in rabbit skeletal muscle

We studied how enhanced skeletal troponin C (sTnC) Ca2+-binding affinity affects cooperative thin filament activation and contraction in single demembranated rabbit psoas fibres. Three sTnC mutants were created and incorporated into skeletal troponin (sTn) for measurement of Ca2+ dissociation, resulting in the following order of rates: wild-type (WT) sTnC-sTn>sTnC(F27W)-sTn>M80Q sTnC-sTn>M80Q sTnCF27W-sTn. Reconstitution of sTnC-extracted fibres increased Ca2+ sensitivity of steady-state force (pCa(50)) by 0.08 for M80Q sTnC, 0.15 for sTnCF27W and 0.32 for M80Q sTnCF27W with minimal loss of slope (nH, degree of cooperativity). Near-neighbour thin filament regulatory unit (RU) interactions were reduced in fibres by incorporating mixtures of WT or mutant sTnC and D28A, D64A sTnC (xxsTnC) that does not bind Ca2+ at N-terminal sites. Reconstitution with sTnC: xxsTnC mixtures to 20% of pre-exchanged maximal force reduced pCa50 by 0.35 for sTnC: xxsTnC, 0.25 for M80Q sTnC: xxsTnC, and 0.10 for M80Q sTnCF27W: xxsTnC. It is interesting that pCa50 increased by approximately 0.1 for M80Q sTnC and approximately 0.3 for M80Q sTnCF27W when near-neighbour RU interactions were reduced; these values are similar in magnitude to those for fibres reconstituted with 100% mutant sTnC. After reconstitution with sTnC: xxsTnC mixtures, nH decreased to a similar value for all mutant sTnCs. Altered sTnC Ca2+-binding properties (M80Q sTnCF27W) did not affect strong crossbridge inhibition by 2,3-butanedione monoxime when near-neighbour thin filament RU interactions were reduced. Together these results suggest increased sTnC Ca2+ affinity strongly influences Ca2+ sensitivity of steady-state force without affecting near-neighbour thin filament RU cooperative activation or the relative contribution of crossbridges versus Ca2+ to thin filament activation.

Sarcomeric determinants of striated muscle relaxation kinetics

Ca2+ is the primary regulator of force generation by cross-bridges in striated muscle activation and relaxation. Relaxation is as necessary as contraction and, while the kinetics of Ca2+-induced force development have been investigated extensively, those of force relaxation have been both studied and understood less well. Knowledge of the molecular mechanisms underlying relaxation kinetics is of special importance for understanding diastolic function and dysfunction of the heart. A number of experimental models, from whole muscle organs and intact muscle fibres down to single myofibrils, have been used to explore the cascade of kinetic events leading to mechanical relaxation. By using isolated myofibrils and fast solution switching techniques we can distinguish the sarcomeric mechanisms of relaxation from those of myoplasmic Ca2+ removal. There is strong evidence that cross-bridge mechanics and kinetics are major determinants of the time course of striated muscle relaxation whilst thin filament inactivation kinetics and cooperative activation of thin filament by cycling, force-generating cross-bridges do not significantly limit the relaxation rate. Results in myofibrils can be explained well by a simple two-state model of the cross-bridge cycle in which the apparent rate of the force generating transition is modulated by fast, Ca2+-dependent equilibration between off- and on-states of actin. Inter-sarcomere dynamics during the final rapid phase of full force relaxation are responsible for deviations from this simple model.

Thin filament near-neighbour regulatory unit interactions affect rabbit skeletal muscle steady-state force-Ca(2+) relations.

The role of cooperative interactions between individual structural regulatory units (SUs) of thin filaments (7 actin monomers : 1 tropomyosin : 1 troponin complex) on steady-state Ca(2+)-activated force was studied. Native troponin C (TnC) was extracted from single, de-membranated rabbit psoas fibres and replaced by mixtures of purified rabbit skeletal TnC (sTnC) and recombinant rabbit sTnC (D27A, D63A), which contains mutations that disrupt Ca(2+) coordination at N-terminal sites I and II (xxsTnC). Control experiments in fibres indicated that, in the absence of Ca(2+), both sTnC and xxsTnC bind with similar apparent affinity to sTnC-extracted thin filaments. Endogenous sTnC-extracted fibres reconstituted with 100 % xxsTnC did not develop Ca(2+)-activated force. In fibres reconstituted with mixtures of sTnC and xxsTnC, maximal Ca(2+)-activated force increased in a greater than linear manner with the fraction of sTnC. This suggests that Ca(2+) binding to functional Tn can spread activation beyond the seven actins of an SU into neighbouring units, and the data suggest that this functional unit (FU) size is up to 10-12 actins. As the number of FUs was decreased, Ca(2+) sensitivity of force (pCa(50)) decreased proportionally. The slope of the force-pCa relation (the Hill coefficient, n(H)) also decreased when the reconstitution mixture contained < 50 % sTnC. With 15 % sTnC in the reconstitution mixture, n(H) was reduced to 1.7 +/- 0.2, compared with 3.8 +/- 0.1 in fibres reconstituted with 100 % sTnC, indicating that most of the cooperative thin filament activation was eliminated. The results suggest that cooperative activation of skeletal muscle fibres occurs primarily through spread of activation to near-neighbour FUs along the thin filament (via head-to-tail tropomyosin interactions).

Thin Filament Cooperativity as a Major Determinant of Shortening Velocity in Skeletal Muscle Fibers

The mechanism underlying the calcium sensitivity of the velocity of shortening of skeletal muscle fibers was investigated using a multiple shortening protocol: within a single contraction, skinned rabbit psoas fibers were made to shorten repetitively under a light load by briefly stretching back to their initial length at regular intervals. At saturating [Ca 2+], the initial fast shortening pattern was repeated reproducibly. At submaximal [Ca 2+], the first shortening consisted of fast and slow phases, but only the slow phase was observed in later shortenings. When the fibers were held isometric after the first shortening, the velocity of the second shortening recovered with time. The recovery paralleled tension redevelopment, implying a close relationship between the velocity and the number of the preexisting force-producing cross-bridges. However, this parallelism was lost as [Ca 2+] was increased. Thus, the velocity was modified in a manner consistent with the cooperative thin filament activation by strong binding cross-bridges and its modulation by calcium. The present results therefore provide evidence that the thin filament cooperativity is primarily responsible for the calcium sensitivity of velocity. The effect of inorganic phosphate to accelerate the slow phase of shortening is also explained in terms of the cooperative activation.

Steady-state [Ca2+]i-force relationship in intact twitching cardiac muscle: direct evidence for modulation by isoproterenol and EMD 53998

We have developed a novel method for measuring steady-state force-[Ca2+]i relations in isolated, membrane-intact rat trabeculae that are microinjected with Fura-2 salt. Twitches are markedly slowed after inhibition of phasic Ca2+ release and uptake from the sarcoplasmic reticulum by addition of cyclopiazonic acid and ryanodine. During relaxation of slowed twitches, force and [Ca2+]i trace a common trajectory in plots of force versus [Ca2+]i, despite very different histories of contraction. The common trajectory thereby provides a high resolution determination of the steady-state relation between force and [Ca2+]i. Using this method, we show that 1 microM isoproterenol, a beta-adrenergic agonist, causes a rightward shift (Hill function K1/2 increased from 0.39 +/- 0.07 microM to 0.82 +/- 0.23 microM, p < 0.02, n = 6) and a decreased slope (nH decreased from 5.4 +/- 1.1 to 4.0 +/- 1.4, p < 0.02) of the steady-state force-[Ca2+]i curve, with no change in maximal force (Fmax = 99.2 +/- 2.2% of control). In contrast, 2 microM EMD 53998, a racemic thiadiazinone derivative, causes a leftward shift (K1/2 decreased from 0.42 +/- 0.02 microM to 0.30 +/- 0.06 microM, p < 0.02, n = 4) with no change in slope of the steady-state force-[Ca2+]i curve, accompanied by a modest increase in maximal force (Fmax = 107.1 +/- 4.6% of control, p < 0.02). To gain mechanistic insight into these modulatory events, we developed a simple model of cooperative thin filament activation that predicts steady-state force-[Ca2+]i relationships. Model analysis suggests that isoproterenol decreases cooperativity arising from nearest-neighbor interactions between regulatory units on the thin filament, without change in the equilibrium constant for Ca2+ binding. In contrast, the effects of EMD 53998 are consistent with an increase in the affinity of strong-binding cross-bridges, without change in either the affinity of troponin C for Ca2+ or cooperative interactions.