Connecting Sarcomere Protein Mutations to Pathogenesis in Cardiomyopathies: The Development of “Disease in a Dish” Models

Recent technological and protocol developments have greatly increased the ability to utilize stem cells transformed into cardiomyocytes as models to study human heart muscle development and how this is affected by disease associated mutations in a variety of sarcomere proteins. In this perspective we provide an overview of these emerging technologies and how they are being used to create better models of ‘disease in a dish’ for both research and screening assays. We also consider the value of these assays as models to explore the seminal processes in initiation of the disease development and the possibility of early interventions

Filament Compliance Influences Cooperative Activation of Thin Filaments and the Dynamics of Force Production in Skeletal Muscle

Striated muscle contraction is a highly cooperative process initiated by Ca2+ binding to the troponin complex, which leads to tropomyosin movement and myosin cross-bridge (XB) formation along thin filaments. Experimental and computational studies suggest skeletal muscle fiber activation is greatly augmented by cooperative interactions between neighboring thin filament regulatory units (RU-RU cooperativity; 1 RU = 7 actin monomers+1 troponin complex+1 tropomyosin molecule). XB binding can also amplify thin filament activation through interactions with RUs (XB-RU cooperativity). Because these interactions occur with a temporal order, they can be considered kinetic forms of cooperativity. Our previous spatially-explicit models illustrated that mechanical forms of cooperativity also exist, arising from XB-induced XB binding (XB-XB cooperativity). These mechanical and kinetic forms of cooperativity are likely coordinated during muscle contraction, but the relative contribution from each of these mechanisms is difficult to separate experimentally. To investigate these contributions we built a multi-filament model of the half sarcomere, allowing RU activation kinetics to vary with the state of neighboring RUs or XBs. Simulations suggest Ca2+ binding to troponin activates a thin filament distance spanning 9 to 11 actins and coupled RU-RU interactions dominate the cooperative force response in skeletal muscle, consistent with measurements from rabbit psoas fibers. XB binding was critical for stabilizing thin filament activation, particularly at submaximal Ca2+ levels, even though XB-RU cooperativity amplified force less than RU-RU cooperativity. Similar to previous studies, XB-XB cooperativity scaled inversely with lattice stiffness, leading to slower rates of force development as stiffness decreased. Including RU-RU and XB-RU cooperativity in this model resulted in the novel prediction that the force-[Ca2+] relationship can vary due to filament and XB compliance. Simulations also suggest kinetic forms of cooperativity occur rapidly and dominate early to get activation, while mechanical forms of cooperativity act more slowly, augmenting XB binding as force continues to develop

Sarcomere Lattice Geometry Influences Cooperative Myosin Binding in Muscle

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

Multiscale modeling of twitch contractions in cardiac trabeculae

© 2021 Mijailovich et al. Understanding the dynamics of a cardiac muscle twitch contraction is complex because it requires a detailed understanding of the kinetic processes of the Ca2+ transient, thin-filament activation, and the myosin-actin cross-bridge chemomechanical cycle. Each of these steps has been well defined individually, but understanding how all three of the processes operate in combination is a far more complex problem. Computational modeling has the potential to provide detailed insight into each of these processes, how the dynamics of each process affect the complexity of contractile behavior, and how perturbations such as mutations in sarcomere proteins affect the complex interactions of all of these processes. The mechanisms involved in relaxation of tension during a cardiac twitch have been particularly difficult to discern due to nonhomogeneous sarcomere lengthening during relaxation. Here we use the multiscale MUSICO platform to model rat trabecular twitches. Validation of computational models is dependent on being able to simulate different experimental datasets, but there has been a paucity of data that can provide all of the required parameters in a single experiment, such as simultaneous measurements of force, intracellular Ca2+ transients, and sarcomere length dynamics. In this study, we used data from different studies collected under similar experimental conditions to provide information for all the required parameters. Our simulations established that twitches either in an isometric sarcomere or in fixed-length, multiple-sarcomere trabeculae replicate the experimental observations if models incorporate a length-tension relationship for the nonlinear series elasticity of muscle preparations and a scheme for thick-filament regulation. The thick-filament regulation assumes an off state in which myosin heads are parked onto the thick-filament backbone and are unable to interact with actin, a state analogous to the super-relaxed state. Including these two mechanisms provided simulations that accurately predict twitch contractions over a range of different conditions

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 time scales from 0.1 – 1000 sec 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 labelled 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 to myofibrils from fast twitch muscle, slow-twitch and cardiac muscles myofibrils require a 10-fold 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

A Stochastic Multiscale Model of Cardiac Thin Filament Activation Using Brownian-Langevin Dynamics.

We use Brownian-Langevin dynamics principles to derive a coarse-graining multiscale myofilament model that can describe the thin-filament activation process during contraction. The model links atomistic molecular simulations of protein-protein interactions in the thin-filament regulatory unit to sarcomere-level activation dynamics. We first calculate the molecular interaction energy between tropomyosin and actin surface using Brownian dynamics simulations. This energy profile is then generalized to account for the observed tropomyosin transitions between its regulatory stable states. The generalized energy landscape then served as a basis for developing a filament-scale model using Langevin dynamics. This integrated analysis, spanning molecular to thin-filament scales, is capable of tracking the events of the tropomyosin conformational changes as it moves over the actin surface. The tropomyosin coil with flexible overlap regions between adjacent tropomyosins is represented in the model as a system of coupled stochastic ordinary differential equations. The proposed multiscale approach provides a more detailed molecular connection between tropomyosin dynamics, the trompomyosin-actin interaction-energy landscape, and the generated force by the sarcomere

Effect of Troponin Ca2+ Binding Properties on the Kinetics of Myofibril Force Initiation and Relaxation

Digitalitzat per Artypla

Contribution of the Myosin Binding Protein C Motif to Functional Effects in Permeabilized Rat Trabeculae

Myosin binding protein C (MyBP-C) is a thick-filament protein that limits cross-bridge cycling rates and reduces myocyte power output. To investigate mechanisms by which MyBP-C affects contraction, we assessed effects of recombinant N-terminal domains of cardiac MyBP-C (cMyBP-C) on contractile properties of permeabilized rat cardiac trabeculae. Here, we show that N-terminal fragments of cMyBP-C that contained the first three immunoglobulin domains of cMyBP-C (i.e., C0, C1, and C2) plus the unique linker sequence termed the MyBP-C “motif” or “m-domain” increased Ca2+ sensitivity of tension and increased rates of tension redevelopment (i.e., ktr) at submaximal levels of Ca2+. At concentrations ≥20 μM, recombinant proteins also activated force in the absence of Ca2+ and inhibited maximum Ca2+-activated force. Recombinant proteins that lacked the combination of C1 and the motif did not affect contractile properties. These results suggest that the C1 domain plus the motif constitute a functional unit of MyBP-C that can activate the thin filament

Mendelian randomization: estimation of inpatient hospital costs attributable to obesity.

BACKGROUND: Mendelian Randomization is a type of instrumental variable (IV) analysis that uses inherited genetic variants as instruments to estimate causal effects attributable to genetic factors. This study aims to estimate the impact of obesity on annual inpatient healthcare costs in the UK using linked data from the UK Biobank and Hospital Episode Statistics (HES). METHODS: UK Biobank data for 482,127 subjects was linked with HES inpatient admission records, and costs were assigned to episodes of care. A two-stage least squares (TSLS) IV model and a TSLS two-part cost model were compared to a naïve regression of inpatient healthcare costs on body mass index (BMI). RESULTS: The naïve analysis of annual cost on continuous BMI predicted an annual cost of £21.61 [95% CI £20.33 - £22.89] greater cost per unit increase in BMI. The TSLS IV model predicted an annual cost of £14.36 [95% CI £0.31 - £28.42] greater cost per unit increase in BMI. Modelled with a binary obesity variable, the naïve analysis predicted that obese subjects incurred £205.53 [95% CI £191.45 - £219.60] greater costs than non-obese subjects. The TSLS model predicted a cost £201.58 [95% CI £4.32 - £398.84] greater for obese subjects compared to non-obese subjects. CONCLUSIONS: The IV models provide evidence for a causal relationship between obesity and higher inpatient healthcare costs. Compared to the naïve models, the binary IV model found a slightly smaller marginal effect of obesity, and the continuous IV model found a slightly smaller marginal effect of a single unit increase in BMI