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The cardiac thin filament is regulated in a Ca2+-dependent manner through conformational changes of troponin and tropomyosin (Tm). It has been generally understood that under conditions of low Ca2+ the inhibitory peptide domain (IP) of troponin I (TnI) binds to actin and holds Tm over the myosin binding sites on actin to prevent crossbridge formation. More recently, evidence that the C-terminal mobile domain (MD) of TnI also binds actin has made for a more complex scenario. This study uses a computational model to investigate the consequences of assuming that TnI regulates Tm movement via two actin-binding domains rather than one. First, a 16-state model of the cardiac thin filament regulatory unit was created with TnI-IP as the sole regulatory domain. Expansion of this to include TnI-MD formed a 24-state model. Comparison of these models showed that assumption of a second actin-binding site allows the individual domains to have a lower affinity for actin than would be required for IP acting alone. Indeed, setting actin affinities of the IP and MD to 25% of that assumed for the IP in the single-site model was sufficient to achieve precisely the same degree of Ca2+ regulation. We also tested the 24-state model's ability to represent steady-state experimental data in the case of disruption of either the IP or MD. We were able to capture qualitative changes in several properties that matched what was seen in the experimental data. Lastly, simulations were run to examine the effect of disruption of the IP or MD on twitch dynamics. Our results suggest that both domains are required to keep diastolic cross-bridge activity to a minimum and accelerate myofilament relaxation. Overall, our analyses support a paradigm in which two domains of TnI bind with moderate affinity to actin, working in tandem to complete Ca2+-dependent regulation of the thin filament.

New findings What is the central question of this study? In the papillary muscle from type I diabetic rats, does diabetes-associated altered ventricular function result from changes of acto-myosin interactions and are these modifications attributable to a possible sarcomere rearrangement? What is the main finding and its importance? For the first time, we showed that type-I diabetes altered sarcomeric ultrastructure, as seen by transmission electron microscopy, consistent with physiological parameters. The diabetic condition induced slower timing parameters, which is compatible with a diastolic dysfunction. At the sarcomeric level, augmented β-myosin heavy chain content and increased sarcomere length and crossbridges' number preserve myocardial stroke and could concur to maintain the ejection fraction. Abstract We investigated whether diabetes-associated altered ventricular function, in a type I diabetes animal model, results from a modification of acto-myosin interactions, through the in vitro recording of left papillary muscle mechanical parameters and examination of sarcomere morphology by transmission electron microscopy (TEM). Experiments were performed on streptozotocin-induced diabetic and age-matched control female Wistar rats. Mechanical isometric and isotonic indexes and timing parameters were determined. Using Huxley's equations, we calculated mechanics, kinetics and energetics of myosin crossbridges. Sarcomere length and A-band length were measured on TEM images. Type I and III collagen and β-myosin heavy chain (MHC) expression were determined by immunoblotting. No variation in resting and developed tension or maximum extent of shortening was evident between groups, but diabetic rats showed lower maximum shortening velocity and prolonged timing parameters. Compared to controls, diabetics also displayed a higher number of crossbridges with lower unitary force. Moreover, no change in type I and III collagen was associated to diabetes, but pathological rats showed a two-fold enhancement of β-MHC content and longer sarcomeres and A-band, detected by ultrastructural morphometry. Overall, these data address whether a preserved systolic function accompanied by an altered diastolic phase results from a recruitment of super-relaxed myosin heads or the phosphorylation of the regulatory light chain site in myosin. Although the early signs of diabetic cardiomyopathy were well expressed, the striking finding of our study was that, in diabetics, sarcomere modification may be a possible compensatory mechanism that preserves systolic function.

Diabetes doubles the risk of developing heart failure (HF). As the prevalence of diabetes grows, so will HF unless the mechanisms connecting these diseases can be identified. Methylglyoxal (MG) is a glycolysis by-product that forms irreversible modifications on lysine and arginine, called glycation. We previously found that myofilament MG glycation causes sarcomere contractile dysfunction and is increased in patients with diabetes and HF. The aim of this study was to discover the molecular mechanisms by which MG glycation of myofilament proteins cause sarcomere dysfunction and to identify therapeutic avenues to compensate. In humans with type 2 diabetes without HF, we found increased glycation of sarcomeric actin compared to non-diabetics and it correlated with decreased calcium sensitivity. Depressed calcium sensitivity is pathogenic for HF, therefore myofilament glycation represents a promising therapeutic target to inhibit the development of HF in diabetics. To identify possible therapeutic targets, we further defined the molecular actions of myofilament glycation. Skinned myocytes exposed to 100 μM MG exhibited decreased calcium sensitivity, maximal calcium-activated force, and crossbridge kinetics. Replicating MG’s functional affects using a computer simulation of sarcomere function predicted simultaneous decreases in tropomyosin’s blocked-to-closed rate transition and crossbridge duty cycle were consistent with all experimental findings. Stopped-flow experiments and ATPase activity confirmed MG decreased the blocked-to-closed transition rate. Currently, no therapeutics target tropomyosin, so as proof-of-principal, we used a n-terminal peptide of myosin-binding protein C, previously shown to alter tropomyosin’s position on actin. C0C2 completely rescued MG-induced calcium desensitization, suggesting a possible treatment for diabetic HF.

Hypertrophic cardiomyopathy (HCM) is an inherited disorder caused primarily by mutations to thick and thinfilament proteins. Although thin filament mutations are less prevalent than their oft-studied thick filament counterparts, they are frequently associated with severe patient phenotypes and can offer important insight into fundamental disease mechanisms. We have performed a detailed study of tropomyosin (TPM1) E192K, a variant of uncertain significance associated with HCM. Molecular dynamics revealed that E192K results in a more flexible TPM1 molecule, which could affect its ability to regulate crossbridges. In vitro motility assays of regulated actin filaments containing TPM1 E192K showed an overall loss of Ca2+ sensitivity. To understand these effects, we used multiscale computational models that suggested a subtle phenotype in which E192K leads to an inability to completely inhibit actin–myosin crossbridge activity at low Ca2+. To assess the physiological impact of the mutation, we generated patient-derived engineered heart tissues expressing E192K. These tissues showed disease features similar to those of the patients, including cellular hypertrophy, hypercontractility, and diastolic dysfunction. We hypothesized that excess residual crossbridge activity could be triggering cellular hypertrophy, even if the overall Ca2+ sensitivity was reduced by E192K. To test this hypothesis, the cardiac myosin–specific inhibitor mavacamten was applied to patient-derived engineered heart tissues for 4 d followed by 24 h of washout. Chronic mavacamten treatment abolished contractile differences between control and TPM1 E192K engineered heart tissues and reversed hypertrophy in cardiomyocytes. These results suggest that the TPM1 E192K mutation triggers cardiomyocyte hypertrophy by permitting excess residual crossbridge activity. These studies also provide direct evidence that myosin inhibition by mavacamten can counteract the hypertrophic effects of mutant tropomyosin.

The cyclic interaction between myosin crossbridges and actin filaments underlies smooth muscle contraction. Phosphorylation of the 20-kDa myosin light chain (MLC20) is a crucial step in activating the crossbridge cycle. Our current understanding of smooth muscle contraction is based on observed correlations among MLC20 phosphorylation, maximal shortening velocity ( Vmax), and isometric force over the time course of contraction. However, during contraction there are changes in the extent of phosphorylation of many additional proteins as well as changes in activation of enzymes associated with the signaling pathways. As a consequence, the mechanical manifestation of muscle contraction is likely to change with time. To simplify the study of these relationships, we measured the mechanical properties of airway smooth muscle at different levels of MLC20 phosphorylation at a fixed time during contraction. A simple correlation emerged when time-dependent variables were fixed. MLC20 phosphorylation was found to be directly and linearly correlated with the active stress, stiffness, and power of the muscle; the observed weak dependence of Vmax on MLC20 phosphorylation could be explained by the presence of an internal load in the muscle preparation. These results can be entirely explained by the Huxley crossbridge model. We conclude that when the influence of time-dependent events during contraction is held constant, the basic crossbridge mechanism in smooth muscle is the same as that in striated muscle.

Movement of the myocardium can modify organ-level cardiac function and its molecular (crossbridge) mechanisms. This motion, which is defined by myocardial strain and strain rate (muscle shortening, lengthening, and the speed of these movements), occurs throughout the cardiac cycle, including during isovolumic periods. This review highlights how the left ventricular myocardium moves throughout the cardiac cycle, how muscle mechanics experiments provide insight into the regulation of forces used to move blood in and out of the left ventricle, and its impact on (and regulation by) crossbridge and sarcomere kinetics. We specifically highlight how muscle mechanics experiments explain how myocardial relaxation is accelerated by lengthening (strain rate) during late systole and isovolumic relaxation, a lengthening which has been measured in human hearts. Advancing and refining both in vivo measurement and ex vivo protocols with physiologic strain and strain rates could reveal important insights into molecular (crossbridge) kinetics. These advances could provide an improvement in both diagnosis and precise treatment of cardiac dysfunction.

This review focuses on thin filament regulatory mechanisms with emphasis on cardiac-specific mechanisms in the three-state model of sarcomere activation and on signaling cascades at the barbed and pointed ends., Our review focuses on sarcomere regulatory mechanisms with a discussion of cardiac-specific modifications to the three-state model of thin filament activation from a blocked to closed to open state. We discuss modulation of these thin filament transitions by Ca2+, by crossbridge interactions, and by thick filament–associated proteins, cardiac myosin–binding protein C (cMyBP-C), cardiac regulatory light chain (cRLC), and titin. Emerging evidence supports the idea that the cooperative activation of the thin filaments despite a single Ca2+ triggering regulatory site on troponin C (cTnC) cannot be considered in isolation of other functional domains of the sarcomere. We discuss long- and short-range interactions among these domains with the regulatory units of thin filaments, including proteins at the barbed end at the Z-disc and the pointed end near the M-band. Important to these discussions is the ever-increasing understanding of the role of cMyBP-C, cRLC, and titin filaments. Detailed knowledge of these control processes is critical to the understanding of mechanisms sustaining physiological cardiac state with varying hemodynamic load, to better defining genetic and acquired cardiac disorders, and to developing targets for therapies at the level of the sarcomeres.

Nonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration. Mammalian cells express up to three isoforms termed NM IIA, B, and C. We used U2OS cells to create CRISPR/Cas9-based knockouts of all three isoforms and analyzed the phenotypes on homogenously coated surfaces, in collagen gels, and on micropatterned substrates. In contrast to homogenously coated surfaces, a structured environment supports a cellular phenotype with invaginated actin arcs even in the absence of NM IIA-induced contractility. A quantitative shape analysis of cells on micropatterns combined with a scale-bridging mathematical model reveals that NM IIA is essential to build up cellular tension during initial stages of force generation, while NM IIB is necessary to elastically stabilize NM IIA-generated tension. A dynamic cell stretch/release experiment in a three-dimensional scaffold confirms these conclusions and in addition reveals a novel role for NM IIC, namely the ability to establish tensional homeostasis.

Aim Altered organisation of the transverse-tubular network is an early pathological event occurring even prior to the onset of heart failure. Such t-tubular remodelling disturbs the synchrony and signalling between membranous and intracellular ion channels, exchangers, receptors and ATPases essential in the dynamics of excitation-contraction coupling, leading to ionic abnormality and mechanical dysfunction in heart disease progression. In this study, we investigated whether a disrupted t-tubular network has a direct effect on cardiac mechano-energetics. Our aim was to understand the fundamental link between t-tubular remodelling and impaired energy metabolism, both of which are characteristics of heart failure. We thus studied healthy tissue preparations in which cellular processes are not altered by any disease event. Methods We exploited the 'formamide-detubulation' technique to acutely disrupt the t-tubular network in rat left-ventricular trabeculae. We assessed the energy utilisation by cellular Ca2+ cycling and by crossbridge cycling, and quantified the change of energy efficiency following detubulation. For these measurements, trabeculae were mounted in a microcalorimeter where force and heat output were simultaneously measured. Results Following structural disorganisation from detubulation, muscle heat output associated with Ca2+ cycling was reduced, indicating impaired intracellular Ca2+ homeostasis. This led to reduced force production and heat output by crossbridge cycling. The reduction in force-length work was not paralleled by proportionate reduction in the heat output and, as such, energy efficiency was reduced. Conclusions These results reveal the direct energetic consequences of disrupted t-tubular network, linking the energy disturbance and the t-tubular remodelling typically observed in heart failure.

X-ray diffraction and tension measurement experiments were conducted on rat left ventricular skinned fibers with or without &ldquo, troponin-T treatment,&rdquo, which exchanges the endogenous troponin T/I/C complex with exogenous troponin-T. These experiments were performed to observe the structural changes in troponin-T within a fiber elicited by contractile crossbridge formation and investigate the abnormality of hypertrophic cardiomyopathy-related troponin-T mutants. The intensity of the troponin reflection at 1/38.5 nm&minus, 1 was decreased significantly by ATP addition after treatment with wild-type or mutant troponin-T, indicating that crossbridge formation affected the conformation of troponin-T. In experiments on cardiac fibers treated with the hypertrophic cardiomyopathy-related mutants E244D- and K247R-troponin-T, treatment with K247R-troponin-T did not recruit contracting actomyosin to a greater extent than wild-type-troponin-T, although a similar drop in the intensity of the troponin reflection occurred. Therefore, the conformational change in K247R-troponin-T was suggested to be unable to fully recruit actomyosin interaction, which may be the cause of cardiomyopathy.

Palmer et al. demonstrate that a stretch-induced enhancement of the myosin crossbridge detachment rate is sufficient to explain the force response of cardiac muscle to a quick stretch and would benefit diastolic function in a beating heart., The force response of cardiac muscle undergoing a quick stretch is conventionally interpreted to represent stretching of attached myosin crossbridges (phase 1) and detachment of these stretched crossbridges at an exponential rate (phase 2), followed by crossbridges reattaching in increased numbers due to an enhanced activation of the thin filament (phases 3 and 4). We propose that, at least in mammalian cardiac muscle, phase 2 instead represents an enhanced detachment rate of myosin crossbridges due to stretch, phase 3 represents the reattachment of those same crossbridges, and phase 4 is a passive-like viscoelastic response with power-law relaxation. To test this idea, we developed a two-state model of crossbridge attachment and detachment. Unitary force was assigned when a crossbridge was attached, and an elastic force was generated when an attached crossbridge was displaced. Attachment rate, f(x), was spatially distributed with a total magnitude f0. Detachment rate was modeled as g(x) = g0 + g1x, where g0 is a constant and g1 indicates sensitivity to displacement. The analytical solution suggested that the exponential decay rate of phase 2 represents (f0 + g0) and the exponential rise rate of phase 3 represents g0. The depth of the nadir between phases 2 and 3 is proportional to g1. We prepared skinned mouse myocardium and applied a 1% stretch under varying concentrations of inorganic phosphate (Pi). The resulting force responses fitted the analytical solution well. The interpretations of phases 2 and 3 were consistent with lower f0 and higher g0 with increasing Pi. This novel scheme of interpreting the force response to a quick stretch does not require enhanced thin-filament activation and suggests that the myosin detachment rate is sensitive to stretch. Furthermore, the enhanced detachment rate is likely not due to the typical detachment mechanism following MgATP binding, but rather before MgADP release, and may involve reversal of the myosin power stroke.

The principle of mechanopharmacology of airway smooth muscle (ASM) is based on the premise that physical agitation, such as pressure oscillation applied to an airway, is able to induce bronchodilation by reducing contractility and softening the cytoskeleton of ASM. Although the underlying mechanism is not entirely clear, there is evidence to suggest that large-amplitude stretches are able to disrupt the actomyosin interaction in the crossbridge cycle and weaken the cytoskeleton in ASM cells. Rho-kinase is known to enhance force generation and strengthen structural integrity of the cytoskeleton during smooth muscle activation and plays a key role in the maintenance of force during prolonged muscle contractions. Synergy in relaxation has been observed when the muscle is subject to oscillatory length change while Rho-kinase is pharmacologically inhibited. In this review, inhibition of Rho-kinase coupled to therapeutic pressure oscillation applied to the airways is explored as a combination treatment for asthma.

Skeletal muscle contains motor units with component myocytes innervated by spinal cord α-motorneurons. The presynaptic terminals release vesicles of the transmitter acetylcholine on stimulation. This combines with postsynaptic nicotinic cholinergic receptors causing their channel opening causing a local endplate potential. If sufficiently large, this causes Na+ channel and therefore action potential activation and propagation through the myocyte surface and transverse tubular membranes. Tubular dihydropyridine receptors detect and transduce this voltage change into ryanodine receptor-mediated sarcoplasmic reticular Ca2 + release. The released Ca2 + combines with troponin triggering actin-myosin interaction resulting in the crossbridge formation and cycling underlying muscle contraction.

Cell walls are barriers found in almost all known bacterial cells. These structures establish a controlled interface between the external environment and vital cellular components. A primary component of cell wall is a highly crosslinked matrix called peptidoglycan (PG). PG crosslinking, carried out by transglycosylases and transpeptidases, is necessary for proper cell wall assembly. Transpeptidases, targets of β-lactam antibiotics, stitch together two neighboring PG stem peptides (acyl-donor and acyl-acceptor strands). We recently described a novel class of cellular PG probes that were processed exclusively as acyl-donor strands. Herein, we have accessed the other half of the transpeptidase reaction by developing probes that are processed exclusively as acyl-acceptor strands. The critical nature of the crossbridge on the PG peptide was demonstrated in live bacterial cells and surprising promiscuity in crossbridge primary sequence was found in various bacterial species. Additionally, acyl-acceptor probes provided insight into how chemical remodeling of the PG crossbridge (e.g., amidation) can modulate crosslinking levels, thus establishing a physiological role of PG structural variations. Together, the acyl-donor and -acceptor probes will provide a versatile platform to interrogate PG crosslinking in physiologically relevant settings.SYNOPSIS TOC

Calcium regulation of cardiac muscle contraction is controlled by the thin-filament proteins troponin and tropomyosin bound to actin. In the absence of calcium, troponin-tropomyosin inhibits myosin-interactions on actin and induces muscle relaxation, whereas the addition of calcium relieves the inhibitory constraint to initiate contraction. Many mutations in thin filament proteins linked to cardiomyopathy appear to disrupt this regulatory switching. Here, we tested perturbations caused by mutant tropomyosins (E40K, DCM; and E62Q, HCM) on intra-filament interactions affecting acto-myosin interactions including those induced further by myosin association. Comparison of wild-type and mutant human α-tropomyosin (Tpm1.1) behavior was carried out using in vitro motility assays and molecular dynamics simulations. Our results show that E62Q tropomyosin destabilizes thin filament off-state function by increasing calcium-sensitivity, but without apparent affect on global tropomyosin structure by modifying coiled-coil rigidity. In contrast, the E40K mutant tropomyosin appears to stabilize the off-state, demonstrates increased tropomyosin flexibility, while also decreasing calcium-sensitivity. In addition, the E40K mutation reduces thin filament velocity at low myosin concentration while the E62Q mutant tropomyosin increases velocity. Corresponding molecular dynamics simulations indicate specific residue interactions that are likely to redefine underlying molecular regulatory mechanisms, which we propose explain the altered contractility evoked by the disease-causing mutations.

KEY POINTS Pulmonary arterial hypertension (PAH) triggers right ventricle (RV) hypertrophy and left ventricle (LV) atrophy, which progressively leads to heart failure. We designed experiments under conditions mimicking those encountered by the heart in vivo that allowed us to investigate whether consequent structural and functional remodelling of the ventricles affects their respective energy efficiencies. We found that peak work output was lower in RV trabeculae from PAH rats due to reduced extent and velocity of shortening. However, their suprabasal enthalpy was unaffected due to increased activation heat, resulting in reduced suprabasal efficiency. There was no effect of PAH on LV suprabasal efficiency. We conclude that the mechanism underlying the reduced energy efficiency of hypertrophied RV tissues is attributable to the increased energy cost of Ca2+ cycling, whereas atrophied LV tissues still maintain normal mechano-energetic performance. ABSTRACT Pulmonary arterial hypertension (PAH) greatly increases the afterload on the right ventricle (RV), triggering RV hypertrophy, which progressively leads to RV failure. In contrast, the disease reduces the passive filling pressure of the left ventricle (LV), resulting in LV atrophy. We investigated whether these distinct structural and functional consequences to the ventricles affect their respective energy efficiencies. We studied trabeculae isolated from both ventricles of Wistar rats with monocrotaline-induced PAH and their respective Control groups. Trabeculae were mounted in a calorimeter at 37°C. While contracting at 5 Hz, they were subjected to stress-length work-loops over a wide range of afterloads. They were subsequently required to undergo a series of isometric contractions at various muscle lengths. In both protocols, stress production, length change and suprabasal heat output were simultaneously measured. We found that RV trabeculae from PAH rats generated higher activation heat, but developed normal active stress. Their peak external work output was lower due to reduced extent and velocity of shortening. Despite lower peak work output, suprabasal enthalpy was unaffected, thereby rendering suprabasal efficiency lower. Crossbridge efficiency, however, was unaffected. In contrast, LV trabeculae from PAH rats maintained normal mechano-energetic performance. Pulmonary arterial hypertension reduces the suprabasal energy efficiency of hypertrophied right ventricular tissues as a consequence of the increased energy cost of Ca2+ cycling.

Activation heat arises from two sources during the contraction of striated muscle. It reflects the metabolic expenditure associated with Ca2+ pumping by the sarcoplasmic reticular Ca2+ ATPase and Ca2+ translocation by the Na+-Ca2+ exchanger coupled to the Na+-K+ ATPase. In cardiac preparations, investigators are constrained to estimate its magnitude by reducing muscle length to the point where macroscopic twitch force vanishes. But this experimental protocol has been criticised since, at zero force, the observed heat may be contaminated by residual cross-bridge cycling activity. To eliminate this concern, the putative thermal contribution from cross-bridge cycling activity must be abolished, at least at minimal muscle length. We achieve this using blebbistatin, a selective inhibitor of myosin II ATPase. Using a microcalorimeter, we measured the force production and heat output, as functions of muscle length, of isolated rat trabeculae from both ventricles contracting isometrically at 5 Hz and at 37°C. In the presence of blebbistatin (15 μmol L−1), active force was zero but heat output remained constant, at all muscle lengths. Activation heat measured in the presence of blebbistatin was not different from that estimated from the intercept of heat-stress relation in its absence. We thus reach two conclusions. First, activation heat is independent of muscle length. Second, residual cross-bridge heat is negligible at zero active force; hence, the intercept of the cardiac heat-force relation provides an estimate of activation heat uncontaminated by crossbridge cycling. Both results resolve long-standing disputes in the literature. This article is protected by copyright. All rights reserved

Key points Actomyosin ATP hydrolysis occurring during muscle contraction releases inorganic phosphate [Pi] in the myoplasm. High [Pi] reduces force and affects force kinetics in skinned muscle fibres at low temperature. These effects decrease at high temperature, raising the question of their importance under physiological conditions. This study provides the first analysis of the effects of Pi on muscle performance in intact mammalian fibres at physiological temperature. Myoplasmic [Pi] was raised by fatiguing the fibres with a series of tetanic contractions. [Pi] increase reduces muscular force mainly by decreasing the force of the single molecular motor, the crossbridge, and alters the crossbridge response to fast length perturbation indicating faster kinetics. These results are in agreement with schemes of actomyosin ATPase and the crossbridge cycle including a low- or no-force state and show that fibre length changes perturb the Pi-sensitive force generation of the crossbridge cycle. Abstract Actomyosin ATP hydrolysis during muscle contraction releases inorganic phosphate, increasing [Pi] in the myoplasm. Experiments in skinned fibres at low temperature (10–12°C) have shown that [Pi] increase depresses isometric force and alters the kinetics of actomyosin interaction. However, the effects of Pi decrease with temperature and this raises the question of the role of Pi under physiological conditions. The present experiments were performed to investigate this point. Intact fibre bundles isolated from the flexor digitorum brevis of C57BL/6 mice were stimulated with a series of tetanic contractions at 1.5 s intervals at 33°C. As show previously the most significant change induced by a bout of contractile activity similar to the initial 10 tetani of the series was an increase of [Pi] without significant Ca2+ or pH changes. Measurements of force, stiffness and responses to fast stretches and releases were therefore made on the 10th tetanus of the series and compared with control. We found that (i) tetanic force at the 10th tetanus was ∼20% smaller than control without a significant decrease of crossbridge stiffness; and (ii) the force recovery following quick stretches and releases was faster than in control. These results indicate that at physiological temperature the increase of [Pi] occurring during early fatigue reduces tetanic force mainly by depressing the individual crossbridge force and accelerating crossbridge kinetics.

A simple model of cardiac muscle was designed for multiscale simulation of heart mechanics. Relaxed cardiac muscle was described as a transversally isotropic hyperelastic material. Active tension caused by actin-myosin crossbridges depends on the ensemble averaged strain of myosin heads bound to actin. Calcium activation was modeled by Ca2+ binding to troponin-C. To account for the dependence of troponin affinity for Ca2+ on myosin heads strongly bound to actin, the kinetics of troponin binding to Ca2+ in the overlap zone of the thin and thick filaments and outside it were separated. The changes in the length of these zones during muscle shortening or lengthening were accounted for explicitly. Simplified version of the model contains only 5 ordinary differential equations (ODE). Model parameters were estimated from a limited set of experiments with skeletal and cardiac muscle. Simulations have shown that model reproduces qualitatively a number of experimental observations: steady-state force-velocity and stiffness-velocity relations; mechanical responses to step changes in muscle length or load; steep Ca2+-tension relationship and its dependence on sarcomere length tension (the Frank-Starling mechanism); tension, shortening and Ca2+-transients in twitch isometric and isotonic contractions, tension development and redevelopment upon fast change in Ca2+ concentration or muscle release followed by re-stretch. We believe that the model can be effectively used for modeling contraction and relaxation of the heart.

The N-terminal extension of human cardiac troponin T (TnT), which modulates myofilament Ca2+ sensitivity, contains several hypertrophic cardiomyopathy (HCM)-causing mutations including S69F. However, the functional consequence of S69F mutation is unknown. The human analog of S69F in rat TnT is L71F (TnTL71F). Because the functional consequences due to structural changes in the N-terminal extension are influenced by the type of myosin heavy chain (MHC) isoform, we hypothesized that the TnTL71F-mediated effect would be differently modulated by α- and β-MHC isoforms. TnTL71F and wild-type rat TnT were reconstituted into de-membranated muscle fibers from normal (α-MHC) and propylthiouracil-treated rat hearts (β-MHC) to measure steady-state and dynamic contractile parameters. The magnitude of the TnTL71F-mediated attenuation of Ca2+-activated maximal tension was greater in α- than in β-MHC fibers. For example, TnTL71F attenuated maximal tension by 31% in α-MHC fibers but only by 10% in β-MHC fibers. Furthermore, TnTL71F reduced myofilament Ca2+ sensitivity by 0.11 pCa units in α-MHC fibers but only by 0.05 pCa units in β-MHC fibers. TnTL71F augmented rate constants of crossbridge recruitment and crossbridge detachment dynamics in α-MHC fibers but not in β-MHC fibers. Collectively, our data demonstrate that TnTL71F induces greater contractile deficits against α-MHC than against β-MHC background.

Tropomyosin, best known for its role in the steric regulation of muscle contraction, polymerizes head-to-tail to form cables localized along the length of both muscle and non-muscle actin-based thin filaments. In skeletal and cardiac muscles, tropomyosin, under the control of troponin and myosin, moves in a cooperative manner between blocked, closed and open positions on filaments, thereby masking and exposing actin-binding sites necessary for myosin crossbridge head interactions. While the coiled-coil signature of tropomyosin appears to be simple, closer inspection reveals surprising structural complexity required to perform its role in steric regulation. For example, component α-helices of coiled coils are typically zippered together along a continuous core hydrophobic stripe. Tropomyosin, however, contains a number of anomalous, functionally controversial, core amino acid residues. We argue that the atypical residues at this interface, including clusters of alanines and a charged aspartate, are required for preshaping tropomyosin to readily fit to the surface of the actin filament, but do so without compromising tropomyosin rigidity once the filament is assembled. Indeed, persistence length measurements of tropomyosin are characteristic of a semi-rigid cable, in this case conducive to cooperative movement on thin filaments. In addition, we also maintain that tropomyosin displays largely unrecognized and residue-specific torsional variance, which is involved in optimizing contacts between actin and tropomyosin on the assembled thin filament. Corresponding twist-induced stiffness may also enhance cooperative translocation of tropomyosin across actin filaments. We conclude that anomalous core residues of tropomyosin facilitate thin filament regulatory behavior in a multifaceted way.

2’-deoxy-ATP (dATP) is a naturally occurring small molecule that has shown promise as a therapeutic because it significantly increases cardiac myocyte force development even at low dATP/ATP ratios. To investigate mechanisms by which dATP alters myosin crossbridge dynamics, we used Brownian dynamics simulations to calculate association rates between actin and ADP- or dADP-bound myosin. These rates were then directly incorporated in a mechanistic Monte Carlo Markov Chain model of cooperative sarcomere contraction. A unique combination of increased powerstroke and detachment rates was required to match experimental steady-state and kinetic data for dATP force production in rat cardiac myocytes when the myosin attachment rate in the model was constrained by the results of a Brownian dynamics simulation. Nearest-neighbor cooperativity was seen to contribute to, but not fully explain, the steep relationship between dATP/ATP ratio and steady-state force-development observed at lower dATP concentrations. Dynamic twitch simulations performed using measured calcium transients as inputs showed that the effects of dATP on the crossbridge alone were not sufficient to explain experimentally observed enhancement of relaxation kinetics by dATP treatment. Hence, dATP may also affect calcium handling even at low concentrations. By enabling the effects of dATP on sarcomere mechanics to be predicted, this multi-scale modelling framework may elucidate the molecular mechanisms by which dATP can have therapeutic effects on cardiac contractile dysfunction.

Sarcomere length dependent activation (LDA) of myocardial force development is the cellular basis underlying the Frank-Starling law of the heart, but it is still elusive how the sarcomeres detect the length changes and convert them into altered activation of thin filament. In this study we investigated how the C-domain of cardiac troponin I (cTnI) functionally and structurally responds to the comprehensive effects of the Ca(2+), crossbridge, and sarcomere length of chemically skinned myocardial preparations. Using our in situ technique which allows for simultaneous measurements of time-resolved FRET and mechanical force of the skinned myocardial preparations, we measured changes in the FRET distance between cTnI(167C) and cTnC(89C), labeled with FRET donor and acceptor, respectively, as a function of [Ca(2+)], crossbridge state and sarcomere length of the skinned muscle preparations. Our results show that [Ca(2+)], cross-bridge feedback and sarcomere length have different effects on the structural transition of the C-domain cTnI. In particular, the interplay between crossbridges and sarcomere length has significant impacts on the functional structural change of the C-domain of cTnI in the relaxed state. These novel observations suggest the importance of the C-domain of cTnI and the dynamic and complex interplay between various components of myofilament in the LDA mechanism.

Stretching of an activated skeletal muscle induces a transient tension increase followed by a period during which the tension remains elevated well above the isometric level at an almost constant value. This excess of tension in response to stretching has been called ‘static tension’ and attributed to an increase in fibre stiffness above the resting value, named ‘static stiffness’. This observation was originally made, by our group, in frog intact muscle fibres and has been confirmed more recently, by us, in mammalian intact fibres. Following stimulation, fibre stiffness starts to increase during the latent period well before crossbridge force generation and it is present throughout the whole contraction in both single twitches and tetani. Static stiffness is dependent on sarcomere length in a different way from crossbridge force and is independent of stretching amplitude and velocity. Static stiffness follows a time course which is distinct from that of active force and very similar to the myoplasmic calcium concentration time course. We therefore hypothesize that static stiffness is due to a calcium-dependent stiffening of a non-crossbridge sarcomere structure, such as the titin filament. According to this hypothesis, titin, in addition to its well-recognized role in determining the muscle passive tension, could have a role during muscle activity.

Microvolt T-wave alternans (MTWA) testing identifies heart failure patients at risk for lethal ventricular arrhythmias at near-resting heart rates (

Mesenchymal stromal cells (MSCs) were obtained from human bone marrow and amplified in cultures supplemented with human platelet lysate. Once semi-confluent, cells were seeded in solid collagen scaffolds that were rapidly colonized by the cells generating a 3D cell scaffold. Here, they acquired a myofibroblast phenotype and when exposed to appropriate chemical stimulus, developed tension and cell shortening, similar to those of striated and smooth muscle cells. Myofibroblasts contained a molecular motor&mdash, the non-muscle myosin type IIA (NMMIIA) whose crossbridge (CB) kinetics are dramatically slow compared with striated and smooth muscle myosins. Huxley&rsquo, s equations were used to determine the molecular mechanical properties of NMMIIA. Thank to the great number of NMMIIA molecules, we determined the statistical mechanics (SM) of MSCs, using the grand canonical ensemble which made it possible to calculate various thermodynamic entities such as the chemical affinity, statistical entropy, internal energy, thermodynamic flow, thermodynamic force, and entropy production rate. The linear relationship observed between the thermodynamic force and the thermodynamic flow allowed to establish that MSC-laden in collagen scaffolds were in a near-equilibrium stationary state (affinity ≪ RT), MSCs were also seeded in solid collagen scaffolds functionalized with the tripeptide Arg-Gly-Asp (RGD). This induced major changes in NMMIIA SM particularly by increasing the rate of entropy production. In conclusion, collagen scaffolds laden with MSCs can be viewed as a non-muscle contractile bioengineered tissue operating in a near-equilibrium linear regime, whose SM could be substantially modified by the RGD peptide.

Muscle contraction is caused by the action of myosin motors within the structural confines of contractile unit arrays. When the force generated by cyclic interactions between myosin crossbridges and actin filaments is greater than the average load shared by the crossbridges, sliding of the actin filaments occurs and the muscle shortens. The shortening velocity as a function of muscle load can be described mathematically by a hyperbola; this characteristic force–velocity relationship stems from stochastic interactions between the crossbridges and the actin filaments. Beyond the actomyosin interaction, there is not yet a unified theory explaining smooth muscle contraction, mainly because the structure of the contractile unit in smooth muscle (akin to the sarcomere in striated muscle) is still undefined. In this review, functional and structural data from airway smooth muscle are analyzed in an engineering approach of quantification and correlation to support a model of the contractile unit with characteristics revealed by mathematical analyses and behavior matched by experimental observation.

An ultimate test of theories of muscle contraction is to reproduce contractile behaviour as a function of the sarcoplasmic concentration of calcium ions. In muscle, the cooperative nature of Ca2+-activation seen with regulated actin in solution is manifest in the steep rise of isometric tension and ATPase rate with [Ca2+], and also in the regulation of transient responses. This chapter surveys the wealth of behaviour observed in striated muscle, fibres, myofibrils and motility assays, before turning to theories of cooperative regulation in fibres. For a single thin filament, a lattice theory of cooperative activation with TmTn units as a continuous flexible chain is developed, and explored computationally with a simple crossbridge model. Spontaneous oscillatory contractions (SPOC) constitute a new state of muscle observed only at low calcium, which can be modelled in terms of enhanced length activation on the descending limb. Finally, we consider direct myosin regulation by its light chains, and whether this mechanism can act cooperatively.

The isometric tetanic tension of skeletal muscle increases with temperature because attached crossbridge states bearing a relatively low force convert to those bearing a higher force. It was previously proposed that the tension-generating step(s) in the crossbridge cycle was highly endothermic and was therefore itself directly targeted by changes in temperature. However, this did not explain why a rapid rise in temperature (a temperature jump) caused a much slower rate of rise of tension than a rapid length step. This led to suggestions that the step targeted by a temperature rise is not the tension-generating step but is an extra step in the attached pathway of the crossbridge cycle, perhaps located on a parallel pathway. This enigma has been a major obstacle to a full understanding of the operation of the crossbridge cycle. We have now used a previously developed mechano-kinetic model of the crossbridge cycle in frog muscle to simulate the temperature dependence of isometric tension and shortening velocity. We allowed all five steps in the cycle to be temperature-sensitive. Models with different starting combinations of enthalpy changes and activation enthalpies for the five steps were refined by downhill simplex runs and scored by their ability to fit experimental data on the temperature dependence of isometric tension and the relationship between force and shortening velocity in frog muscle. We conclude that the first tension-generating step may be weakly endothermic and that the rise of tension with temperature is largely driven by the preceding two strongly endothermic steps of ATP hydrolysis and attachment of M.ADP.Pi to actin. The refined model gave a reasonable fit to the available experimental data and after a temperature jump the overall rate of tension rise was much slower than after a length step as observed experimentally. The findings aid our understanding of the crossbridge cycle by showing that it may not be necessary to include an additional temperature-sensitive step.

When skeletal muscles are stretched during activation in the absence of myosin-actin interactions, the force increases significantly. The force remains elevated throughout the activation period. The mechanism behind this non-crossbridge force, referred to as static tension, is unknown and generates debate in the literature. It has been suggested that the static tension is caused by Ca(2+)-induced changes in the properties of titin molecules that happens during activation and stretch, but a comprehensive evaluation of such possibility is still lacking. This paper reviews the general characteristics of the static tension, and evaluates the proposed mechanism by which titin may change the force upon stretch. Evidence is presented suggesting that an increase in intracellular Ca(2+) concentration leads to Ca(2+) binding to the PEVK region of titin. Such binding increases titin stiffness, which increases the overall sarcomere stiffness and causes the static tension. If this form of Ca(2+)-induced increase in titin stiffness is confirmed in future studies, it may have large implications for understating of the basic mechanisms of muscle contraction.

A. Huxley's equations were used to determine the mechanical properties of muscle myosin II (MII) at the molecular level, as well as the probability of the occurrence of the different stages in the actin-myosin cycle. It was then possible to use the formalism of statistical mechanics with the grand canonical ensemble to calculate numerous thermodynamic parameters such as entropy, internal energy, affinity, thermodynamic flow, thermodynamic force, and entropy production rate. This allows us to compare the thermodynamic parameters of a non-muscle contractile system, such as the normal human placenta, with those of different striated skeletal muscles (soleus and extensor digitalis longus) as well as the heart muscle and smooth muscles (trachea and uterus) in the rat. In the human placental tissues, it was observed that the kinetics of the actin-myosin crossbridges were considerably slow compared with those of smooth and striated muscular systems. The entropy production rate was also particularly low in the human placental tissues, as compared with that observed in smooth and striated muscular systems. This is partly due to the low thermodynamic flow found in the human placental tissues. However, the unitary force of non-muscle myosin (NMII) generated by each crossbridge cycle in the myofibroblasts of the human placental tissues was similar in magnitude to that of MII in the myocytes of both smooth and striated muscle cells. Statistical mechanics represents a powerful tool for studying the thermodynamics of all contractile muscle and non-muscle systems.

Background and aims Although studies on the relation between arrhythmias and the action potential duration (APD) have been carried out, most of them are based only on electrophysiological factors of the heart and lack experiments that consider cardiac mechanical and electromechanical characteristics. Therefore, we conducted this study to clarify the relevance of the shortening of APD of a cell in relation to the mechanical contraction activity of the heart and the associated risk of arrhythmia. Methods The human ventricular model used in this study has two dynamic characteristics: electrophysiological conduction and mechanical contraction. The model simulating electrophysiological characteristics was consisted of lumped parameter circuit that can mimic the phenomenon of ion exchange through the cell membrane of myocyte and consisted of 214,319 tetrahedral finite elements. In contrast, the model simulating mechanical contraction characteristics was constructed to mimic cardiac contraction by means of the crossbridge of a myofilament and consisted of 14,720 hermite-based finite elements to represent a natural 3D curve of the cardiac surface. First, we performed a single cell simulation and the electrophysiological simulation according to the change of the APD by changing the electrical conductivity of the I Ks channel. Thus, we confirmed the correlation between APD and intracellular Ca2+ concentration. Then, we compared mechanical response through mechanical simulation using Ca2+ data from electrical simulation. Results The APD and the sum of the intracellular Ca2+ concentrations showed a positive correlation. The shortened APD reduced the conduction wavelength of ventricular cells by shortening the plateau and early repolarization in myocardial cells. The decrease in APD reduced ventricular pumping efficiency by more than 60% as compared with the normal group (normal conditions). This change is caused by the decline of ventricular output owing to reduced ATP consumption during the crossbridge of myofilaments and decreased tension. Conclusion The shortening of APD owing to increased electrical conductivity of a protein channel on myocardial cells likely decreases the wavelength and the pumping efficiency of the ventricles. Additionally, it may increase tissue sensitivity to ventricular fibrillation, including reentry, and cause symptoms such as dyspnea and dizziness.

A new contraction model of cardiac muscle was developed by combining previously described biochemical and biophysical models. The biochemical component of the new contraction model represents events in the presence of Ca2+–crossbridge attachment and power stroke following inorganic phosphate release, detachment evoked by the replacement of ADP by ATP, ATP hydrolysis, and recovery stroke. The biophysical component focuses on Ca2+ activation and force (F b) development assuming an equivalent crossbridge. The new model faithfully incorporates the major characteristics of the biochemical and biophysical models, such as F b activation by transient Ca2+ ([Ca2+]–F b), [Ca2+]–ATP hydrolysis relations, sarcomere length–F b, and F b recovery after jumps in length under the isometric mode and upon sarcomere shortening after a rapid release of mechanical load under the isotonic mode together with the load–velocity relationship. ATP consumption was obtained for all responses. When incorporated in a ventricular cell model, the contraction model was found to share approximately 60% of the total ATP usage in the cell model.

Experimental data from human cardiac myocytes at body temperature is crucial for a quantitative understanding of clinically relevant cardiac function and development of whole-organ computational models. However, such experimental data is currently very limited. Specifically, important measurements to characterize changes in tension development in human cardiomyocytes that occur with perturbations in cell length are not available. To address this deficiency, in this study we present an experimental data set collected from skinned human cardiac myocytes, including the passive and viscoelastic properties of isolated myocytes, the steady-state force calcium relationship at different sarcomere lengths, and changes in tension following a rapid increase or decrease in length, and after constant velocity shortening. This data set is, to our knowledge, the first characterization of length and velocity-dependence of tension generation in human skinned cardiac myocytes at body temperature. We use this data to develop a computational model of contraction and passive viscoelasticity in human myocytes. Our model includes troponin C kinetics, tropomyosin kinetics, a three-state crossbridge model that accounts for the distortion of crossbridges, and the cellular viscoelastic response. Each component is parametrized using our experimental data collected in human cardiomyocytes at body temperature. Furthermore we are able to confirm that properties of length-dependent activation at 37 °C are similar to other species, with a shift in calcium sensitivity and increase in maximum tension. We revise our model of tension generation in the skinned isolated myocyte to replicate reported tension traces generated in intact muscle during isometric tension, to provide a model of human tension generation for multi-scale simulations. This process requires changes to calcium sensitivity, cooperativity, and crossbridge transition rates. We apply this model within multi-scale simulations of biventricular cardiac function and further refine the parametrization within the whole organ context, based on obtaining a healthy ejection fraction. This process reveals that crossbridge cycling rates differ between skinned myocytes and intact myocytes.

In experiments on active muscle, we examined the tension decline and its temperature sensitivity at the onset of ramp shortening and at a range of velocities. A segment (∼1.5 mm long) of a skinned muscle fibre isolated from rabbit psoas muscle was held isometric (sarcomere length ∼2.5µm) at 8-9 °C, maximally Ca-activated and a ramp shortening applied. The tension decline with a ramp shortening showed an early decrease of slope (the P1 transition) followed by a slower decrease in slope (the P2 transition) to the steady (isotonic) force. The tension level at the initial P1 transition and the time t1 to that transition decreased as the velocity is increased; the length change L1 to this transition increased with shortening velocity to a steady value of ∼8 nm / half-sarcomere. A small rapid temperature jump (3-4 °C

We test the hypothesis that cytosolic inorganic phosphate (Pi) can account for the contraction‐induced reductions in twitch duration which impair summation and cause force to decline (sag) during unfused tetanic contractions of fast‐twitch muscle. A five‐state model of crossbridge cycling was used to simulate twitch and unfused tetanic contractions. As Pi concentration ([Pi]) was increased from 0 to 30 mmol·L−1, twitch duration decreased, with progressive reductions in sensitivity to Pi as [Pi] was increased. When unfused tetani were simulated with rising [Pi], sag was most pronounced when initial [Pi] was low, and when the magnitude of [Pi] increase was large. Fast‐twitch extensor digitorum longus (EDL) muscles (sag‐prone, typically low basal [Pi]) and slow‐twitch soleus muscles (sag‐resistant, typically high basal [Pi]) were isolated from 14 female C57BL/6 mice. Muscles were sequentially incubated in solutions containing either glucose or pyruvate to create typical and low Pi environments, respectively. Twitch duration was greater (P glucose; P glucose; P

The functional significance of the molecular swivel at the head-to-tail overlapping ends of contiguous tropomyosin (Tm) dimers in striated muscle is unknown. Contractile measurements were made in muscle fibers from transgenic (TG) mouse hearts that expressed a mutant α-Tm (TmH276N). We also reconstituted mouse cardiac troponin T (McTnT) N-terminal deletion mutants, McTnT1–44Δ and McTnT45–74Δ, into muscle fibers from TmH276N. For controls, we used the wild-type (WT) McTnT because altered effects could be correlated with the mutant forms of McTnT. TmH276N slowed crossbridge (XB) detachment rate (g) by 19%. McTnT1–44Δ attenuated Ca2+-activated maximal tension against TmWT (36%) and TmH276N (38%), but sped g only against TmH276N by 35%. The rate of tension redevelopment decreased (17%) only in McTnT1–44Δ + TmH276N fibers. McTnT45–74Δ attenuated tension (19%) and myofilament Ca2+ sensitivity (pCa50=5.93 vs. 6.00 in the control fibers) against TmH276N, but not against TmWT background. Thus, altered XB cycling kinetics decreased the fraction of strongly bound XBs in McTnT1–44Δ + TmH276N fibers, whereas diminished thin-filament cooperativity attenuated tension in McTnT45–74Δ + TmH276N fibers. In summary, our study is the first to show that the interplay between the N terminus of cTnT and the overlapping ends of contiguous Tm effectuates different states of Tm on the actin filament. —Mamidi, R., Michael, J. J., Muthuchamy, M., Chandra, M. Interplay between the overlapping ends of tropomyosin and the N terminus of cardiac troponin T affects tropomyosin states on actin.

Cardiac muscle contraction is activated via the single Ca(2+)-binding site (site II) in the N-domain of troponin C (cTnC). The two Ca(2+)/Mg(2+) binding sites in the C-domain of cTnC (sites III and IV) have been considered to play a purely structural role in anchoring cTnC to the thin filament. However, several recent discoveries suggest a possible role of this domain in contractile regulation. The green tea polyphenol (-)-epigallocatechin 3-gallate (EGCg), which binds specifically to the C-domain of cTnC, reduces cardiac myofilament Ca(2+) sensitivity along with maximum force and acto-myosin ATPase activity. We have determined the effect of EGCg on Ca(2+) and Mg(2+) binding to the C-domain of cTnC. In the absence of Mg(2+) there was no significant effect of EGCg on the Ca(2+)-cTnC affinity. Surprisingly, in the presence of Mg(2+) EGCg caused an increase in Ca(2+) affinity for sites III and IV of cTnC. However, in the absence of Ca(2+) the addition of EGCg caused a significant reduction in Mg(2+)-cTnC affinity. This reduction is presumably responsible for the increase in Ca(2+)-cTnC affinity produced by EGCg in the presence of Mg(2+). We propose that the inhibitory effect of EGCg on myofilament Ca(2+) activation may be related to an enhanced Ca(2+)-Mg(2+)exchange at sites III and IV of cTnC, which might reduce the myosin crossbridge dependent component of thin filament activation.

Protein kinase A (PKA) phosphorylation of myofibrillar proteins constitutes an important pathway for β-adrenergic modulation of cardiac contractility. In myofilaments PKA targets troponin I (cTnI), myosin binding protein-C (cMyBP-C) and titin. We studied how this affects the sarcomere length (SL) dependence of force-pCa relations in demembranated cardiac muscle. To distinguish cTnI from cMyBP-C/titin phosphorylation effects on the force-pCa relationship, endogenous troponin (Tn) was exchanged in rat ventricular trabeculae with either wild-type (WT) Tn, non-phosphorylatable cTnI (S23/24A) Tn or phosphomimetic cTnI (S23/24D) Tn. PKA cannot phosphorylate either cTnI S23/24 variant, leaving cMyBP-C/titin as PKA targets. Force was measured at 2.3 and 2.0 μm SL. Decreasing SL reduced maximal force (F(max)) and Ca(2+) sensitivity of force (pCa(50)) similarly with WT and S23/24A trabeculae. PKA treatment of WT and S23/24A trabeculae reduced pCa(50) at 2.3 but not at 2.0 μm SL, thus eliminating the SL dependence of pCa(50). In contrast, S23/24D trabeculae reduced pCa(50) at both SL values, primarily at 2.3 μm, also eliminating SL dependence of pCa(50). Subsequent PKA treatment moderately reduced pCa(50) at both SLs. At each SL, F(max) was unaffected by either Tn exchange and/or PKA treatment. Low-angle X-ray diffraction was performed to determine whether pCa(50) shifts were associated with changes in myofilament spacing (d(1,0)) or thick-thin filament interaction. PKA increased d(1,0) slightly under all conditions. The ratios of the integrated intensities of the equatorial X-ray reflections (I(1,1)/I(1,0)) indicate that PKA treatment increased crossbridge proximity to thin filaments under all conditions. The results suggest that phosphorylation by PKA of either cTnI or cMyBP-C/titin independently reduces the pCa(50) preferentially at long SL, possibly through reduced availability of thin filament binding sites (cTnI) or altered crossbridge recruitment (cMyBP-C/titin). Preferential reduction of pCa(50) at long SL may not reduce cardiac output during periods of high metabolic demand because of increased intracellular Ca(2+) during β-adrenergic stimulation.

Many studies have demonstrated that depressed myofilament Ca2+ sensitivity is common to dilated cardiomyopathy (DCM) in humans. However, it remains unclear whether a single determinant — such as myofilament Ca2+ sensitivity — is sufficient to characterize all cases of DCM because the severity of disease varies widely with a given mutation. Because dynamic features dominate in the heart muscle, alterations in dynamic contractile parameters may offer better insight on the molecular mechanisms that underlie disparate effects of DCM mutations on cardiac phenotypes. Dynamic features are dominated by myofilament cooperativity that stem from different sources. One such source is the strong tropomyosin binding region in troponin T (TnT), which is known to modulate crossbridge (XB) recruitment dynamics in a myosin heavy chain (MHC)-dependent manner. Therefore, we hypothesized that the effects of DCM-linked mutations in TnT on contractile dynamics would be differently modulated by α- and β-MHC. After reconstitution with the mouse TnT equivalent (TnTR134W) of the human DCM mutation (R131W), we measured dynamic contractile parameters in detergent-skinned cardiac muscle fiber bundles from normal (α-MHC) and transgenic mice (β-MHC). TnTR134W significantly attenuated the rate constants of tension redevelopment, XB recruitment dynamics, XB distortion dynamics, and the magnitude of length-mediated XB recruitment only in α-MHC fiber bundles. TnTR134W decreased myofilament Ca2+ sensitivity to a greater extent in α-MHC (0.14 pCa units) than in β-MHC fiber bundles (0.08 pCa units). Thus, our data demonstrate that TnTR134W induces a more severe DCM-like contractile phenotype against α-MHC than against β-MHC background.

Fast myocardial relaxation is important in vivo to reduce left ventricular pressure and promote efficient filling after cardiac contraction and ejection. Afterload is theorized to be a modifier of relaxation, but afterload reduction does not appear to help patients with diastolic dysfunction or Heart Failure with preserved Ejection Fraction. We hypothesized that a fast stretch, not afterload, modifies the relaxation rate, and that this relationship is modified by the giant elastic protein titin. A rat model carrying a spontaneous mutation in the RNA binding motif-20 (RBM20) results in long titin isoforms. Cardiac trabeculae from wild-type and RBM20 rats were dissected, paced at 0.5 Hz, and stretched to Lo, the length at which maximum isometric twitch force was developed. Isotonic load clamps maintained a specified afterload in experimental twitches by shortening and (if needed) relengthening the muscles using feedback control. Identical protocols were performed at reduced preload and two temperature conditions (37°C and 25°C). We determined that myocardial relaxation rate (1/tau) was modified by fast stretch (strain rate) just prior to relaxation; preventing stretch eliminated the inverse relationship between afterload and relaxation rate. Computational modeling indicates that fast stretch enhances crossbridge detachment and that passive tension compensates for some of the active force reduction during stretch. Stretch of 1 length•s -1 causes a 240% increase in relaxation rate (54.6 s -1 vs 22.5 s -1 ) at 37°C. Relaxation rate was preload independent and was slower but more responsive to stretch at 25°C. Trabeculae from long-titin mutant rats were not statistically different than control animals, but trend (p

The effect of tethering molecules in 3D space on bimolecular binding kinetics is rarely addressed and occasionally incorporated in models of cell motility. The simplest system to quantitatively determine this effect is in the 3D sarcomere lattice of the striated muscle where the tethered myosin can only bind to a relatively few available actin sites, positioned within a limited range of the thermal movement of myosin head. A multiscale Monte Carlo software platform MUSICO takes into account the exact number of unbound myosin molecules interacting only with unoccupied actin sites in their proximity and provides quantitative predictions of muscle response at the macroscopic scale. As such MUSICO is well suited to determine the effect of the distortion of interacting molecules on biochemical reactions, i.e. modulation of state transition rates in the actomyosin cycle by the imposed geometrical constraints of tethered myosin molecules. The simulations provide the distribution of bound sites, preservation of species, and most importantly the departure in nanoscopic behavior of tethered myosin molecules from unconstrained myosin interactions with actin. The MUSICO simulations with the simplest actomyosin cycles show large differences in predicted force velocity curves and during early force recovery phase after step in change in length comparing to all mass-action models. The origin of the differences in predicted muscle response is rooted in the differences the fluxes of myosin binding and corresponding instantaneous crossbridge distributions. Consequently a new approach showed that fitting of the experiments require significantly different binding rates than used in mass-action models and, therefore, to correctly describe the biochemical reactions of tethered molecules and the interaction energetics. The knowledge gained by this analysis can be used for development of new mass-action and other models that can implicitly incorporate this phenomenon via correction factors.

Based on a detailed crossbridge model for individual myosin II motors, we systematically study the influence of mechanical load and adenosine triphosphate (ATP) concentration on small myosin II ensembles made from different isoforms. For skeletal and smooth muscle myosin II, which are often used in actomyosin gels that reconstitute cell contractility, fast forward movement is restricted to a small region of phase space with low mechanical load and high ATP concentration, which is also characterized by frequent ensemble detachment. At high load, these ensembles are stalled or move backwards, but forward motion can be restored by decreasing ATP concentration. In contrast, small ensembles of nonmuscle myosin II isoforms, which are found in the cytoskeleton of nonmuscle cells, are hardly affected by ATP concentration due to the slow kinetics of the bound states. For all isoforms, the thermodynamic efficiency of ensemble movement increases with decreasing ATP concentration, but this effect is weaker for the nonmuscle myosin II isoforms., Comment: 18 pages, 5 figures

We tested the proposition that linear length dependence of twitch duration underlies the well-characterised linear dependence of oxygen consumption (V(O(2)) ) on pressure–volume area (PVA) in the heart. By way of experimental simplification, we reduced the problem from three dimensions to one by substituting cardiac trabeculae for the classically investigated whole-heart. This allowed adoption of stress–length area (SLA) as a surrogate for PVA, and heat as a proxy for V(O(2)) . Heat and stress (force per cross-sectional area), at a range of muscle lengths and at both 1 mM and 2 mM [Ca(2+)](o), were recorded from continuously superfused rat right-ventricular trabeculae undergoing fixed-end contractions. The heat–SLA relations of trabeculae (reported here, for the first time) are linear. Twitch duration increases monotonically (but not strictly linearly) with muscle length. We probed the cellular mechanisms of this phenomenon by determining: (i) the length dependence of the duration of the Ca(2+) transient, (ii) the length dependence of the rate of force redevelopment following a length impulse (an index of Ca(2+) binding to troponin-C), (iii) the effect on the simulated time course of the twitch of progressive deletion of length and Ca(2+)-dependent mechanisms of crossbridge cooperativity, using a detailed mathematical model of the crossbridge cycle, and (iv) the conditions required to achieve these multiple length dependencies, using a greatly simplified model of twitch mechano-energetics. From the results of these four independent investigations, we infer that the linearity of the heat–SLA relation (and, by analogy, the V(O(2))–PVA relation) is remarkably robust in the face of departures from linearity of length-dependent twitch duration.

The aim of the present study was to examine the temperature dependence of active force in lengthening and shortening muscle. Experiments were done, in vitro, on bundles of intact fibres (fibre length L(0) ~2 mm; sarcomere length ~2.5 μm) isolated from a rat fast muscle (flexor hallucis brevis) and a ramp length change of 5-7% L(0) was applied on the plateau of an isometric tetanic contraction. Ramp lengthening increased and ramp shortening decreased the muscle tension to new approximately steady levels in a velocity-dependent way. The isometric tension and the lower steady tension reached at a given shortening velocity, increased with warming from 10 to 35 °C and the relation between tension and reciprocal absolute temperature was sigmoidal. However, the tension-temperature curve of shortening muscle was sharper and shifted to higher temperature with increased velocity. In contrast, the enhanced steady tension during lengthening at a given velocity was largely temperature-insensitive within the same temperature range; we hypothesize that the tension-temperature curve may be shifted to lower temperatures in lengthening muscle. Consequently, when normalised to the isometric tension at each temperature, the tension during lengthening at a given velocity decreased exponentially with increase of temperature. The residual force enhancement that remains after ramp lengthening showed a similar behaviour and was markedly reduced in warming from 10 to 35 °C. The findings are consistent with the thesis that active force generation in muscle is endothermic and strain-sensitive; during shortening with a faster crossbridge cycle it becomes more pronounced, but during lengthening it becomes depressed as the cycle slows in a velocity-dependent way. The residual force enhancement may be caused by the same process in addition to non-crossbridge mechanism(s).

Voltage-dependent ion channels are crucial for generation and propagation of electrical activity in biological systems. The primary mechanism for voltage transduction in these proteins involves the movement of a voltage-sensing domain (D), which opens a gate located on the cytoplasmic side. A distinct conformational change in the selectivity filter near the extracellular side has been implicated in slow inactivation gating, which is important for spike frequency adaptation in neural circuits. However, it remains an open question whether gating transitions in the selectivity filter region are also actuated by voltage sensors. Here, we examine conformational coupling between each of the four voltage sensors and the outer pore of a eukaryotic voltage-dependent sodium channel. The voltage sensors of these sodium channels are not structurally symmetric and exhibit functional specialization. To track the conformational rearrangements of individual voltage-sensing domains, we recorded domain-specific gating pore currents. Our data show that, of the four voltage sensors, only the domain IV voltage sensor is coupled to the conformation of the selectivity filter region of the sodium channel. Trapping the outer pore in a particular conformation with a high-affinity toxin or disulphide crossbridge impedes the return of this voltage sensor to its resting conformation. Our findings directly establish that, in addition to the canonical electromechanical coupling between voltage sensor and inner pore gates of a sodium channel, gating transitions in the selectivity filter region are also coupled to the movement of a voltage sensor. Furthermore, our results also imply that the voltage sensor of domain IV is unique in this linkage and in the ability to initiate slow inactivation in sodium channels.

Actin stress fibers (SFs) play essential roles in cellular contractile force generation and resulting adhesion regulation. Although contractile properties of whole cells have been extensively studied, those intrinsic to individual SFs remain unknown. Here we isolate functional SFs from cells and directly measure the relationship between the shortening velocity and contractile forces generated by individual SFs. These isolated SFs are associated with major structural proteins typical for SFs present within intact cells, which include phosphorylated myosin regulatory light chain (MRLC) required for actin-myosin crossbridge cycling. Thus, contractile shortening of the structurally and functionally non-impaired SFs can be induced in vitro upon application of adenosine triphosphate. We then characterize their contractile properties using functionalized microneedles for physical manipulation and force measurement. SFs shorten quickly in the absence of external loading, whereas, interestingly, the shortening velocity is dramatically decreased by more than two orders of magnitude upon application of a small tensile load. These results show a highly steep shortening velocity-load relationship that is distinct from the hyperbolic Hill relation of skeletal myofibrils, which has been assumed in other studies to be valid for SFs as well because these two have similar sarcomeric patterns along their lengths.