Cross-bridge Research Articles

Small Molecule RPI-194 Stabilizes Activated Troponin to Increase the Calcium Sensitivity of Striated Muscle Contraction.

Small molecule cardiac troponin activators could potentially enhance cardiac muscle contraction in the treatment of systolic heart failure. We designed a small molecule, RPI-194, to bind cardiac/slow skeletal muscle troponin (Cardiac muscle and slow skeletal muscle share a common isoform of the troponin C subunit.) Using solution NMR and stopped flow fluorescence spectroscopy, we determined that RPI-194 binds to cardiac troponin with a dissociation constant KD of 6–24 μM, stabilizing the activated complex between troponin C and the switch region of troponin I. The interaction between RPI-194 and troponin C is weak (KD 311 μM) in the absence of the switch region. RPI-194 acts as a calcium sensitizer, shifting the pCa50 of isometric contraction from 6.28 to 6.99 in mouse slow skeletal muscle fibers and from 5.68 to 5.96 in skinned cardiac trabeculae at 100 μM concentration. There is also some cross-reactivity with fast skeletal muscle fibers (pCa50 increases from 6.27 to 6.52). In the slack test performed on the same skinned skeletal muscle fibers, RPI-194 slowed the velocity of unloaded shortening at saturating calcium concentrations, suggesting that it slows the rate of actin-myosin cross-bridge cycling under these conditions. However, RPI-194 had no effect on the ATPase activity of purified actin-myosin. In isolated unloaded mouse cardiomyocytes, RPI-194 markedly decreased the velocity and amplitude of contractions. In contrast, cardiac function was preserved in mouse isolated perfused working hearts. In summary, the novel troponin activator RPI-194 acts as a calcium sensitizer in all striated muscle types. Surprisingly, it also slows the velocity of unloaded contraction, but the cause and significance of this is uncertain at this time. RPI-194 represents a new class of non-specific troponin activator that could potentially be used either to enhance cardiac muscle contractility in the setting of systolic heart failure or to enhance skeletal muscle contraction in neuromuscular disorders.

On 'The content of troponin, tropomyosin, actin, and myosin in rabbit skeletal muscle myofibrils' by James D. Potter

After the discovery of troponin by Ebashi almost sixty years ago the field of striated muscle regulation has made significant progress. In the 1970's the nascent troponin field gained momentum, including contributions by James D. Potter who established the stoichiometry of contractile proteins in the myofibril (Arch Biochem Biophys. 1974 Jun; 162(2):436-41. https://doi.org/10.1016/0003-9861(7490202-1)). This opened the door to refinement of competing models that described possible thick filament configurations. This study suggested the presence of one myosin per cross bridge and provided accurate calculations of the molar ratios of each protein - myosin: actin: tropomyosin: troponin T: troponin I: troponin C.

Fast stretching of skeletal muscle fibres abolishes residual force enhancement.

The steady-state isometric force of a muscle after active stretching is greater than the steady-state force for a purely isometric contraction at the same length and activation level. The mechanisms underlying this property, termed residual force enhancement (rFE), remain unknown. When myofibrils are actively stretched while cross-bridge cycling is inhibited, rFE is substantially reduced, suggesting that cross-bridge cycling is essential to produce rFE. Our purpose was to further investigate the role of cross-bridge cycling in rFE by investigating whether fast stretching that causes cross-bridge slipping is associated with a loss of rFE. Skinned fibre bundles from rabbit psoas muscles were stretched slowly (0.08 µms-1) or rapidly (800 µms-1) while activated, from an average sarcomere length of 2.4 to 3.2 µm. Force was enhanced by 38±4% (mean±s.e.m) after the slow stretches but was not enhanced after the fast stretches, suggesting that proper cross-bridge cycling is required to produce rFE.

The Impact of Varying Caffeine‐Induced Contractile Activity on Metabolic Enzyme Activity

Exercise intensity is widely known to differentially affect skeletal muscle metabolic approaches, with a general understanding that short duration, high intensity exercise is associated with an increased reliance on anaerobic glycolysis, while long duration, low to moderate intensity exercise is associated with a greater reliance on aerobic processes. In cell culture, caffeine is a commonly used method to pharmaceutically stimulate contractile activity in skeletal muscle cells; upon exposure to caffeine, ryanodine receptors are stimulated to release calcium, thereby allowing crossbridge cycling and subsequent contractile activity to occur. This technique for exercising muscle cells in culture offers a controlled, experimental platform to study the effect of contractile activity on isolated muscle cells; presumably, varying doses of caffeine should mimic varying levels of exercise intensity, though the degree to which caffeine dose impacts metabolic shifts has yet to be fully elucidated. Here, we exposed C2C12 cells, an immortalized line of mouse skeletal muscle cells, to varying levels of caffeine supplementation throughout myotube differentiation in an attempt to recapitulate metabolic signatures consistent with varying exercise intensities. Following standard proliferation protocols, mature myotubes were differentiated for 7 days. On differentiation days 4‐7, experimental cohorts of cells were exposed to either a low dosage (10mM) or a high dosage (40 mM) of caffeine‐supplemented differentiation media for 20 minutes daily. All cells were harvested for protein on differentiation day seven. Protein concentration across cohorts was measured using a Bradford protein assay. Metabolic changes were characterized by running functional enzyme assays for lactate dehydrogenase (LDH) and citrate synthase (CS) to compare anaerobic and aerobic changes, respectively. Preliminary data suggest that, relative to controls, cells exposed to 10mM caffeine have a decrease in LDH activity, and a trend toward increased CS activity. Conversely, cells exposed to 40mM caffeine show no change in LDH and a trend toward decreased CS activity. Collectively, these preliminary data suggest that C2C12 cells exposed to low doses of caffeine simulating low intensity exercise may have less reliance on anaerobic metabolism with a potential greater reliance on aerobic metabolism, while those exposed to high doses of caffeine simulating high intensity exercise may have a lesser reliance on aerobic metabolism. While more research needs to be done to characterize the precise impact of varying caffeine dosages on C2C12 cells, these initial findings are consistent with previously established trends in exercise intensity and metabolism.

Muscle Glycogen in Elite Soccer - A Perspective on the Implication for Performance, Fatigue, and Recovery.

Based on extrapolation of current trends in modern soccer, physiological loading has increased markedly, and the game will continue to become even more demanding in the future, which will exacerbate fatigue at the end of a game and between games. Soccer is a glycogen consuming activity due to its high-intensity intermittent nature, and muscle glycogen is a key factor associated with fatigue late in a game, as well as in determining recovery after a game or an intense training session. Low glycogen in individual muscle fibers and subcellular compartments in the muscle cell is likely to negatively affect several essential steps in the excitation-contraction coupling such as action potential propagation, calcium handling and cross-bridge cycling through reductions in muscle ATP which are suggested sites of muscle function impairment inducing muscle fatigue. Recovery of physical performance and muscle glycogen after a soccer game is a slow process, which challenges the reality in modern elite soccer with increased game and training frequency and physiological loading. We suggest a markedly higher prioritization of fitness training modalities, nutritional approaches and general recovery strategies that optimizes muscle glycogen storage prior to games and training sessions. Also, the soccer community including the governing bodies of the sport must acknowledge and plan according to the high and increasing demands of the modern game, as well as the consequences this has on fatigue and recovery. These aspects are paramount to consider in the planning of training and games, as well as in the process of structuring soccer tournaments and developing competitive regulations in the future to optimize performance and player health.

Identification of Phosphorylation and Other Post-Translational Modifications in the Central C4C5 Domains of Murine Cardiac Myosin Binding Protein C.

Cardiac myosin binding protein C (cMyBPC) is a critical multidomain protein that modulates myosin cross bridge behavior and cardiac contractility. cMyBPC is principally regulated by phosphorylation of the residues within the M-domain of its N-terminus. However, not much is known about the phosphorylation or other post-translational modification (PTM) landscape of the central C4C5 domains. In this study, the presence of phosphorylation outside the M-domain was confirmed in vivo using mouse models expressing cMyBPC with nonphosphorylatable serine (S) to alanine substitutions. Purified recombinant mouse C4C5 domain constructs were incubated with 13 different kinases, and samples from the 6 strongest kinases were chosen for mass spectrometry analysis. A total of 26 unique phosphorylated peptides were found, representing 13 different phosphorylation sites including 10 novel sites. Parallel reaction monitoring and subsequent mutagenesis experiments revealed that the S690 site (UniProtKB O70468) was the predominant target of PKA and PKG1. We also report 6 acetylation and 7 ubiquitination sites not previously described in the literature. These PTMs demonstrate the possibility of additional layers of regulation and potential importance of the central domains of cMyBPC in cardiac health and disease. Data are available via ProteomeXchange with identifier PXD031262.

Driving Utah's Rivers: Working Water in the West

Driving Utah's Rivers: Working Water in the West

A new active contraction model for the myocardium using a modified hill model

This study develops a new hybrid active contraction model for myocardial dynamics abstracted from sarcomere by combining the phenomenologically active-stress based Hill model and the micro-structurally motivated active strain approach. This new model consists of a passive branch and a parallel active branch that consists of a serial passive element for active tension transmission and a contractile unit for active tension development. This rheology represents an additive decomposition of the total stress into a passive and active response. The active stress is formulated following the active strain approach based on the sliding filament theory by multiplicatively decomposing the stretch of the contractile element into a fictitious and an active part. The length-dependence and force-velocity are further incorporated in the active strain. We estimate the passive stiffness of the serial passive element using literature data, which is 250 kPa, then the active stress is computed from the serial passive element in the active branch because of its force transmission structure. This one-dimensional contraction model is further generalized to three dimensions for modelling myocardial dynamics. Our results demonstrate that the proposed active contraction model has a high descriptive capability for various experiments, including both isometric and isotonic contraction compared to existing active strain approaches. We also show that it can simulate physiologically accurate cardiac dynamics in humans. The excellent agreement with experimental data and a local sensitivity study highlight the importance of length-dependence and force-velocity in the active strain approach. Our results further show that there exists a tight interaction between the length-dependence and force-velocity relationships. This new hybrid model serves as a step forward in personalized cardiac modelling using an active-strain based contraction model and has the potential to understand the multi-scale coupling in active contraction according to the sliding filament theory.

Integrative Computational Modeling of Cardiomyocyte Calcium Handling and Cardiac Arrhythmias: Current Status and Future Challenges.

Cardiomyocyte calcium-handling is the key mediator of cardiac excitation-contraction coupling. In the healthy heart, calcium controls both electrical impulse propagation and myofilament cross-bridge cycling, providing synchronous and adequate contraction of cardiac muscles. However, calcium-handling abnormalities are increasingly implicated as a cause of cardiac arrhythmias. Due to the complex, dynamic and localized interactions between calcium and other molecules within a cardiomyocyte, it remains experimentally challenging to study the exact contributions of calcium-handling abnormalities to arrhythmogenesis. Therefore, multiscale computational modeling is increasingly being used together with laboratory experiments to unravel the exact mechanisms of calcium-mediated arrhythmogenesis. This article describes various examples of how integrative computational modeling makes it possible to unravel the arrhythmogenic consequences of alterations to cardiac calcium handling at subcellular, cellular and tissue levels, and discusses the future challenges on the integration and interpretation of such computational data.

Virgin Passive Colon Biomechanics and a Literature Review of Active Contraction Constitutive Models

The objective of this paper is to present our findings on the biomechanical aspects of the virgin passive anisotropic hyperelasticity of the porcine colon based on equibiaxial tensile experiments. Firstly, the characterization of the intestine tissues is discussed for a nearly incompressible hyperelastic fiber-reinforced Holzapfel–Gasser–Ogden constitutive model in virgin passive loading conditions. The stability of the evaluated material parameters is checked for the polyconvexity of the adopted strain energy function using positive eigenvalue constraints of the Hessian matrix with MATLAB. The constitutive material description of the intestine with two collagen fibers in the submucosal and muscular layer each has been implemented in the FORTRAN platform of the commercial finite element software LS-DYNA, and two equibiaxial tensile simulations are presented to validate the results with the optical strain images obtained from the experiments. Furthermore, this paper also reviews the existing models of the active smooth muscle cells, but these models have not been computationally studied here. The review part shows that the constitutive models originally developed for the active contraction of skeletal muscle based on Hill’s three-element model, Murphy’s four-state cross-bridge chemical kinetic model and Huxley’s sliding-filament hypothesis, which are mainly used for arteries, are appropriate for numerical contraction numerical analysis of the large intestine.

Molecular Events of the Crossbridge Cycle Reflected in the Force-Velocity Relationship of Activated Muscle.

Muscles convert chemical energy to mechanical work. Mechanical performance of a muscle is often assessed by the muscle’s ability to shorten and generate power over a range of loads or forces, characterized by the force–velocity and force–power relationships. The hyperbolic force–velocity relationship of muscle, for a long time, has been regarded as a pure empirical description of the force–velocity data. Connections between mechanical manifestation in terms of force–velocity properties and the kinetics of the crossbridge cycle have only been established recently. In this review, we describe how the model of Huxley’s crossbridge kinetics can be transformed to the hyperbolic Hill equation, and link the changes in force–velocity properties to molecular events within the crossbridge cycle driven by ATP hydrolysis. This allows us to reinterpret some findings from previous studies on experimental interventions that altered the force–velocity relationship and gain further insight into the molecular mechanisms of muscle contraction under physiological and pathophysiological conditions.

2-Deoxy-ATP improves ventricular function via combined effects on myosin recruitment from the super-relaxed state, crossbridge cycling and calcium dynamics in healthy and failing conditions

2-deoxy-ATP (dATP) has been shown to enhance cardiac contractility by acting on myosin to increase the rate of crossbridge binding and cycling. An increased rate of Ca2+ transient decay has also been observed with elevated dATP. However, it is unclear how dATP affects feedback between sarcomere force and Ca2+ dynamics, and to what degree these cellular level effects can explain experimentally observed improvements in ventricular function with elevated dATP. Further, low concentrations of dATP have been shown to significantly improve cardiomyocyte and ventricular function, but the mechanisms behind this are not well understood.

Fast stretching of single skeletal muscle fibers abolishes residual force enhancement

It has been shown that the steady-state isometric force of a muscle after active stretching is greater than the steady-state force for a purely isometric contraction at the same length and activation level. This property of skeletal muscle has been called residual force enhancement (rFE) (Abbott & Aubert, 1952). Despite half a century of study, the mechanisms underlying rFE remain unknown. It has been shown that when myofibrils were actively stretched while cross-bridges were not allowed to cycle, rFE was substantially reduced, suggesting that cross-bridge cycling is necessary to induce rFE (Leonard & Herzog, 2010; Powers et al., 2014).

Modeling force redevelopment as a response to thin filament activation time

Force redevelopment follows a characteristic time course after an isometrically contracting muscle fiber is shortened and then re-stretched. Common explanations for the rate of force redevelopment account for cooperative interactions produced by mass action binding of myosin molecules to the thin filament. However, a competing hypothesis is that mechanical force of the stretch increases the odds that a segment of thin filament is available for interactions with cycling crossbridges during a given interval of time.

2'-Deoxy-ATP release myosin heads from the super relaxed state in permeabilized porcine myocardium

The nucleotide 2-deoxy-ATP (dATP) is a naturally occurring energy substrate for myosin that enhances cross-bridge binding and cycling kinetics. In relaxed rodent cardiac and skeletal muscle, dATP repositions myosin heads away from the thick filament backbone towards thin filaments, but the effects of dATP on the myocardium from large mammals has been much less explored. Here we titrated permeabilized porcine myocardium in relaxing solution with a series of dATP concentrations ranging from 0 to 100% of the total nucleotide pool and examined myosin and sarcomere structure using small-angle X-ray diffraction.

Multiscale simulations of the effects of 2’-deoxy-ATP and a myosin mutation on actomyosin interactions

The actin-myosin cross-bridge cycle catalyzes the hydrolysis of ATP, leading to cardiomyocyte contraction. 2'-deoxy-ATP (dATP) is a myosin activator, which increases contractile force in cardiomyocytes even at relatively low concentrations of the adenine nucleotide pool. dATP has therefore been proposed as a therapeutic for heart failure with reduced ejection fraction. Previous studies have demonstrated that, compared with ATP, dATP induces conformational and electrostatic changes in myosin that increase the association rate of myosin to actin.

Machine learning model predicts the effects of tropomyosin rigidity on cardiac muscle contraction

Tropomyosin (Tm) oscillates periodically between three equilibrium states over the surface of actin filament during the cardiac thin filament activation process. This motion has shown to be essential in regulating the access of Myosin heads to actin binding sites, hence the cross-bridge cycling dynamics. Since the trajectory landscape of Tm motions depends critically on its micromechanical characteristics represented primarily by its stiffness or rigidity. It is important to study the effects of Tm stiffness variations on the sarcomere contraction.

Thick and thin filament effectors of cardiac myofibril contraction detected in a troponin c-based high-throughput fluorescence lifetime assay

Cardiac muscle contraction is initiated by Ca2+-induced structural changes of the thin filaments to permit cross-bridge formation. Ca2+ binding to cardiac troponin C (TnC) on actin permits myosin binding and ATP binding to myosin allows for cross-bridge cycling during contraction. Site-directed spectroscopic probes attached to TnC at position T53C have been useful to determine structure-function relationships and Ca2+ binding kinetics in thin filaments and myofibrils by monitoring fluorescence intensity.

Evidence for novel phosphorylation sites in the central C4-C5 domains of murine cardiac myosin binding protein C

Cardiac myosin binding protein C (cMyBPC) is a key multi-domain protein in myosin cross bridge formation and cardiac contractility. It is principally regulated by phosphorylation of the residues within the M-domain in the N-terminus, which is essential for proper function. In fact, cardiac hypertrophy and heart failure have been associated with reduced overall cMyBPC phosphorylation levels. However, not much is known about the phosphorylation landscape of the enigmatic central C4-C5 domains of cMyBPC.

Exploring the super-relaxed state of myosin in myofibrils from fast-twitch, slow-twitch, and cardiac muscle

Muscle myosin heads, in the absence of actin, have been shown to exist in two states, the relaxed (turnover ∼0.05 s−1) and super-relaxed states (SRX, 0.005 s−1) using a simple fluorescent ATP chase assay (Hooijman, P. et al (2011) Biophys. J.100, 1969–1976). Studies have normally used purified proteins, myosin filaments, or muscle fibers. Here we use muscle myofibrils, which retain most of the ancillary proteins and 3-D architecture of muscle and can be used with rapid mixing methods. Recording timescales from 0.1 to 1000 s provides a precise measure of the two populations of myosin heads present in relaxed myofibrils. We demonstrate that the population of SRX states is formed from rigor cross bridges within 0.2 s of relaxing with fluorescently labeled ATP, and the population of SRX states is relatively constant over the temperature range of 5 °C–30 °C. The SRX population is enhanced in the presence of mavacamten and reduced in the presence of deoxy-ATP. Compared with myofibrils from fast-twitch muscle, slow-twitch muscle, and cardiac muscles, myofibrils require a tenfold lower concentration of mavacamten to be effective, and mavacamten induced a larger increase in the population of the SRX state. Mavacamten is less effective, however, at stabilizing the SRX state at physiological temperatures than at 5 °C. These assays require small quantities of myofibrils, making them suitable for studies of model organism muscles, human biopsies, or human-derived iPSCs.