Cross-bridge Binding Research Articles

Can Strain Dependent Inhibition of Cross-Bridge Binding Explain Shifts in Optimum Muscle Length?

Skeletal muscle force is generated by cross-bridge interactions between the overlapping contractile proteins, actin and myosin. The geometry of this overlap gives us the force-length relationship in which maximum isometric force is generated at an intermediate, optimum, length. However, the force-length relationship is not constant; optimum length increases with decreasing muscle activation. This effect is not predicted from actin-myosin overlap. Here we present evidence that this activation-dependent shift in optimum length may be due to a series compliance within muscles. As muscles generate force during fixed-end contractions, fibers shorten against series compliance until forces equilibrate and they become isometric. Shortening against series-compliance is proportional to activation, and creates conditions under which shortening-induced force depression may suppress full force development. Greater shortening will result in greater force depression. Hence, optimum length may decrease as activation rises due to greater fiber shortening. We discuss explanations of such history dependence, giving a review of previously proposed processes and suggesting a novel mechanistic explanation for the most likely candidate process based on tropomyosin kinetics. We suggest this mechanism could change the relationship between actin-myosin overlap and cross-bridge binding potential, not only depressing force at any given length, but also altering the relationship between force and length. This would have major consequences for our understanding of in vivo muscle performance.

A Numerical Study of Scalable Cardiac Electro-Mechanical Solvers on HPC Architectures.

We introduce and study some scalable domain decomposition preconditioners for cardiac electro-mechanical 3D simulations on parallel HPC (High Performance Computing) architectures. The electro-mechanical model of the cardiac tissue is composed of four coupled sub-models: (1) the static finite elasticity equations for the transversely isotropic deformation of the cardiac tissue; (2) the active tension model describing the dynamics of the intracellular calcium, cross-bridge binding and myofilament tension; (3) the anisotropic Bidomain model describing the evolution of the intra- and extra-cellular potentials in the deforming cardiac tissue; and (4) the ionic membrane model describing the dynamics of ionic currents, gating variables, ionic concentrations and stretch-activated channels. This strongly coupled electro-mechanical model is discretized in time with a splitting semi-implicit technique and in space with isoparametric finite elements. The resulting scalable parallel solver is based on Multilevel Additive Schwarz preconditioners for the solution of the Bidomain system and on BDDC preconditioned Newton-Krylov solvers for the non-linear finite elasticity system. The results of several 3D parallel simulations show the scalability of both linear and non-linear solvers and their application to the study of both physiological excitation-contraction cardiac dynamics and re-entrant waves in the presence of different mechano-electrical feedbacks.

Fropofol decreases force development in cardiac muscle.

Supranormal contractile properties are frequently associated with cardiac diseases. Anesthetic agents, including propofol, can depress myocardial contraction. We tested the hypothesis that fropofol, a propofol derivative, reduces force development in cardiac muscles via inhibition of cross-bridge cycling and may therefore have therapeutic potential. Force and intracellular Ca2+ concentration ([Ca2+]i) transients of rat trabecular muscles were determined. Myofilament ATPase, actin-activated myosin ATPase, and velocity of actin filaments propelled by myosin were also measured. Fropofol dose dependently decreased force without altering [Ca2+]i in normal and pressure-induced hypertrophied-hypercontractile muscles. Similarly, fropofol depressed maximum Ca2+-activated force ( Fmax) and increased the [Ca2+]i required for 50% of Fmax (Ca50) at steady state without affecting the Hill coefficient in both intact and skinned cardiac fibers. The drug also depressed cardiac myofibrillar and actin-activated myosin ATPase activity. In vitro actin sliding velocity was significantly reduced when fropofol was introduced during rigor binding of cross-bridges. The data suggest that the depressing effects of fropofol on cardiac contractility are likely to be related to direct targeting of actomyosin interactions. From a clinical standpoint, these findings are particularly significant, given that fropofol is a nonanesthetic small molecule that decreases myocardial contractility specifically and thus may be useful in the treatment of hypercontractile cardiac disorders.-Ren, X., Schmidt, W., Huang, Y., Lu, H., Liu, W., Bu, W., Eckenhoff, R., Cammarato, A., Gao, W. D. Fropofol decreases force development in cardiac muscle.

Effects of cross-bridge compliance on the force-velocity relationship and muscle power output.

Muscles produce force and power by utilizing chemical energy through ATP hydrolysis. During concentric contractions (shortening), muscles generate less force compared to isometric contractions, but consume greater amounts of energy as shortening velocity increases. Conversely, more force is generated and less energy is consumed during eccentric muscle contractions (lengthening). This relationship between force, energy use, and the velocity of contraction has important implications for understanding muscle efficiency, but the molecular mechanisms underlying this behavior remain poorly understood. Here we used spatially-explicit, multi-filament models of Ca2+-regulated force production within a half-sarcomere to simulate how force production, energy utilization, and the number of bound cross-bridges are affected by dynamic changes in sarcomere length. These computational simulations show that cross-bridge binding increased during slow-velocity concentric and eccentric contractions, compared to isometric contractions. Over the full ranges of velocities that we simulated, cross-bridge cycling and energy utilization (i.e. ATPase rates) increased during shortening, and decreased during lengthening. These findings are consistent with the Fenn effect, but arise from a complicated relationship between velocity-dependent cross-bridge recruitment and cross-bridge cycling kinetics. We also investigated how force production, power output, and energy utilization varied with cross-bridge and myofilament compliance, which is impossible to address under typical experimental conditions. These important simulations show that increasing cross-bridge compliance resulted in greater cross-bridge binding and ATPase activity, but less force was generated per cross-bridge and throughout the sarcomere. These data indicate that the efficiency of force production decreases in a velocity-dependent manner, and that this behavior is sensitive to cross-bridge compliance. In contrast, significant effects of myofilament compliance on force production were only observed during isometric contractions, suggesting that changes in myofilament compliance may not influence power output during non-isometric contractions as greatly as changes in cross-bridge compliance. These findings advance our understanding of how cross-bridge and myofilament properties underlie velocity-dependent changes in contractile efficiency during muscle movement.

A missense mutation within the C6-domain of cMyBP-C results in cardiac enlargement and depressed ventricular function

Cardiac myosin-binding protein-C (cMyBP-C) plays a key role in determining the force and kinetics of myocardial contraction, primarily by binding to either myosin subfragment-2 or actin or both. By modulating the probability of myosin cross-bridge binding to actin, cMyBP-C normally acts to govern the kinetics of force development and relaxation. To determine the effects on cardiac morphology and function due to a novel cMyBP-C W792R missense mutation associated with clinical hypertrophic cardiomyopathy in humans. We generated cMyBP-C-W792R heterozygous and homozygous knock-in mice to investigate the structural and functional phenotypes associated with a missense mutation within the C6-domain of cMyBP-C. Compared to wild-type, homozygous W792R knock-in hearts exhibited significant ventricular and atrial dilation. While the W792R mutant protein was expressed at equivalent levels as in wild-type mice, the extent of phosphorylation of the mutant cMyBP-C protein was markedly reduced. Measurements of steady-state force and cross-bridge cycling kinetics showed that homozygous cMyBP-C W792R skinned myocardium exhibited a significant increase in the Ca2+-sensitivity of force and accelerated cross-bridge cycling kinetics (ktr) at maximal levels of Ca2+-activation, compared to WT myocardium. Cardiac function, as assessed by transthoracic echocardiography, revealed depressed indices of systolic and diastolic ventricular function in the homozygous W792R KI mice. These results demonstrate that a mutation within the mid-region of cMyBP-C can disrupt the dynamic regulation between myosin and actin that is normally governed by cMyBP-C, thereby leading to aberrant cardiac enlargement and commensurate diminution of in vivo ventricular function.

Ablation of cMyBP-C eliminates the activation-dependence of unloaded shortening velocity at low levels of Ca2 + activation

At low levels of Ca2+ activation, the time-course of unloaded shortening velocity (Vo) in striated muscle is biphasic, i.e., shortening is comprised of an initial high-velocity phase and a subsequent low-velocity phase. The respective velocities of both the fast and slow phases are known to scale with the level of activation, culminating in a single high-velocity phase at saturating Ca2+ (i.e., Vmax). The molecular mechanism underlying the activation-dependence of Vo most likely involves a shortening-induced cooperative inactivation of the thin filament that results in reduced numbers of strongly-bound cross-bridges, giving rise to the slow phase of unloaded shortening. Cardiac myosin binding protein-C (cMyBP-C) is believed to modulate the cooperative activation of the thin filament by binding to myosin and/or actin and thereby regulating the probability of cross-bridge binding to actin. To test the idea that cMyBP-C contributes, at least in part, to the activation-dependence of unloaded shortening velocity, we measured Vo in skinned trabeculae isolated from wild-type (WT), cTnI-Ala2 (i.e., Ser23,24Ala) and cTnI-Ala2 x cMyBP-C-/- mice. During maximal activation, Vmax was monophasic and not significantly different among the three groups. However, at low levels of activation, biphasic shortening was observed in both the WT and cTnI-Ala2 myocardium, but only the high-velocity phase was observed in the cTnI-Ala2 x cMyBP-C-/- myocardium. Treatment of WT and cTnI-Ala2 trabeculae with PKA accelerated both the low- and high-velocity phases of shortening during submaximal activation, but did not eliminate the low-velocity phase. Thus, phosphorylation of cMyBP-C but not cTnI was required to evoke these effects. The results can be explained in terms of a model in which cMyBP-C regulates the cooperative binding of myosin cross-bridges to actin with the degree of cooperative propagation dependent on the phosphorylation state of cMyBP-C.

Functional significance of C-terminal mobile domain of cardiac troponin I

Ca2+-regulation of cardiac contractility is mediated through the troponin complex, which comprises three subunits: cTnC, cTnI, and cTnT. As intracellular [Ca2+] increases, cTnI reduces its binding interactions with actin to primarily interact with cTnC, thereby enabling contraction. A portion of this regulatory switching involves the mobile domain of cTnI (cTnI-MD), the role of which in muscle contractility is still elusive. To study the functional significance of cTnI-MD, we engineered two cTnI constructs in which the MD was truncated to various extents: cTnI(1–167) and cTnI(1–193). These truncations were exchanged for endogenous cTnI in skinned rat papillary muscle fibers, and their influence on Ca2+-activated contraction and cross-bridge cycling kinetics was assessed at short (1.9 μm) and long (2.2 μm) sarcomere lengths (SLs). Our results show that the cTnI(1–167) truncation diminished the SL-induced increase in Ca2+-sensitivity of contraction, but not the SL-dependent increase in maximal tension, suggesting an uncoupling between the thin and thick filament contributions to length dependent activation. Compared to cTnI(WT), both truncations displayed greater Ca2+-sensitivity and faster cross-bridge attachment rates at both SLs. Furthermore, cTnI(1–167) slowed MgADP release rate and enhanced cross-bridge binding. Our findings imply that cTnI-MD truncations affect the blocked-to closed-state transition(s) and destabilize the closed-state position of tropomyosin.

Molecular mechanisms underlying deoxy-ADP.Pi activation of pre-powerstroke myosin.

Myosin activation is a viable approach to treat systolic heart failure. We previously demonstrated that striated muscle myosin is a promiscuous ATPase that can use most nucleoside triphosphates as energy substrates for contraction. When 2-deoxy ATP (dATP) is used, it acts as a myosin activator, enhancing cross-bridge binding and cycling. In vivo, we have demonstrated that elevated dATP levels increase basal cardiac function and rescues function of infarcted rodent and pig hearts. Here we investigate the molecular mechanism underlying this physiological effect. We show with molecular dynamics simulations that the binding of dADP.Pi (dATP hydrolysis products) to myosin alters the structure and dynamics of the nucleotide binding pocket, myosin cleft conformation, and actin binding sites, which collectively yield a myosin conformation that we predict favors weak, electrostatic binding to actin. In vitro motility assays at high ionic strength were conducted to test this prediction and we found that dATP increased motility. These results highlight alterations to myosin that enhance cross-bridge formation and reveal a potential mechanism that may underlie dATP-induced improvements in cardiac function.

Joint influence of transmural heterogeneities and wall deformation on cardiac bioelectrical activity: A simulation study

The aim of this work is to investigate, by means of numerical simulations, the influence of myocardial deformation due to muscle contraction and relaxation on the cardiac repolarization process in presence of transmural intrinsic action potential duration (APD) heterogeneities. The three-dimensional electromechanical model considered consists of the following four coupled components: the quasi-static transversely isotropic finite elasticity equations for the deformation of the cardiac tissue; the active tension model for the intracellular calcium dynamics and cross-bridge binding; the anisotropic Bidomain model for the electrical current flow through the deforming cardiac tissue; the membrane model of ventricular myocytes, including stretch-activated channels. The numerical simulations are based on our finite element parallel solver, which employs Multilevel Additive Schwarz preconditioners for the solution of the discretized Bidomain equations and Newton-Krylov methods for the solution of the discretized non-linear finite elasticity equations. Our findings show that: (i) the presence of intrinsic transmural cellular APD heterogeneities is not fully masked by electrotonic current flow or by the presence of the mechanical deformation; (ii) despite the presence of transmural APD heterogeneities, the recovery process follows the activation sequence and there is no significant transmural repolarization gradient; (iii) with or without transmural APD heterogeneities, epicardial electrograms always display the same wave shape and discordance between the polarity of QRS complex and T-wave; (iv) the main effects of the mechanical deformation are an increase of the dispersion of repolarization time and APD, when computed over the total cardiac domain and over the endo- and epicardial surfaces, while there is a slight decrease along the transmural direction.

Increased Titin Compliance Reduced Length-Dependent Contraction and Slowed Cross-Bridge Kinetics in Skinned Myocardial Strips from Rbm (20ΔRRM) Mice.

Titin is a giant protein spanning from the Z-disk to the M-band of the cardiac sarcomere. In the I-band titin acts as a molecular spring, contributing to passive mechanical characteristics of the myocardium throughout a heartbeat. RNA Binding Motif Protein 20 (RBM20) is required for normal titin splicing, and its absence or altered function leads to greater expression of a very large, more compliant N2BA titin isoform in Rbm20 homozygous mice (Rbm20ΔRRM) compared to wild-type mice (WT) that almost exclusively express the stiffer N2B titin isoform. Prior studies using Rbm20ΔRRM animals have shown that increased titin compliance compromises muscle ultrastructure and attenuates the Frank-Starling relationship. Although previous computational simulations of muscle contraction suggested that increasing compliance of the sarcomere slows the rate of tension development and prolongs cross-bridge attachment, none of the reported effects of Rbm20ΔRRM on myocardial function have been attributed to changes in cross-bridge cycling kinetics. To test the relationship between increased sarcomere compliance and cross-bridge kinetics, we used stochastic length-perturbation analysis in Ca2+-activated, skinned papillary muscle strips from Rbm20ΔRRM and WT mice. We found increasing titin compliance depressed maximal tension, decreased Ca2+-sensitivity of the tension-pCa relationship, and slowed myosin detachment rate in myocardium from Rbm20ΔRRM vs. WT mice. As sarcomere length increased from 1.9 to 2.2 μm, length-dependent activation of contraction was eliminated in the Rbm20ΔRRM myocardium, even though myosin MgADP release rate decreased ~20% to prolong strong cross-bridge binding at longer sarcomere length. These data suggest that increasing N2BA expression may alter cardiac performance in a length-dependent manner, showing greater deficits in tension production and slower cross-bridge kinetics at longer sarcomere length. This study also supports the idea that passive mechanical characteristics of the myocardium influence ensemble cross-bridge behavior and maintenance of tension generation throughout the sarcomere.

A cross-bridge based model of force depression: Can a single modification address both transient and steady-state behaviors?

Force depression (FD), the reduction of isometric force following active shortening, is a phenomenon of skeletal muscle that has received significant attention in biomechanical and physiological literature, yet the mechanisms underlying FD remain unknown. Recent experiments identified a slower rate of force redevelopment with increasing amounts of steady-state FD, suggesting that FD may be caused, at least in part, by a decrease in cross-bridge binding rate (Corr and Herzog, 2005; Koppes et al., 2014). Herein, we develop a cross-bridge based model of FD in which the binding rate function, f, decreases with the mechanical work performed during shortening. This modification incorporates a direct relationship between steady-state FD and muscle mechanical work (Corr and Herzog, 2005; Herzog et al., 2000; Kosterina et al., 2008), and is consistent with a proposed mechanism attributing FD to stress-induced inhibition of cross-bridge attachments (Herzog, 1998; Maréchal and Plaghki, 1979). Thus, for an increase in mechanical work, the model should predict a slower force redevelopment (decreased attachment rate) to a more depressed steady-state force (fewer attached cross-bridges), and a reduction in contractile element stiffness (Ford et al., 1981). We hypothesized that since this modification affects the cross-bridge kinetics, a corresponding model would be able to account for both transient and steady-state FD behaviors. Comparisons to prior experiments (Corr and Herzog, 2005; Herzog et al., 2000; Kosterina et al., 2008) show that both steady-state and transient aspects of FD, as well as the relationship of FD with respect to speed and amplitude of shortening, are well captured by this model. Thus, this relatively simple cross-bridge based model of FD lends support to a mechanism involving the inhibition of cross-bridge binding, and indicates that cross-bridge kinetics may play a critical role in FD.

Effect of Active Shortening and Stretching on Lattice Spacing and Cross-Bridge Binding in Skinned Muscle Fibres

When a tetanized skeletal muscle is actively shortened or stretched the resulting steady-state force is different from the purely isometric force at the corresponding length. Active shortening reduces steady-state force (termed force depression-FD), while active lengthening increases steady-state force (termed force enhancement-FE). According to the cross-bridge theory, FD and FE can occur if myofilament lattice spacing was modified after active shortening and stretching leading to a change in the proportion of attached cross-bridges, or if the kinetics of cross-bridges were modified leading to a change in the proportion of attached cross-bridges and/or the average force per cross-bridge. Here, we determined the effect of active shortening and stretching on the myofilament lattice spacing and the proximity of the myosin heads to the thin filaments using small angle X-ray diffraction.Lattice spacing (d1,0) was calculated from the spacing between the 1,0 and 1,1 equatorial reflections. The ratio of the intensities of the 1,1 and 1,0 equatorial reflections (I1,1:I1,0) was used as an estimate of the degree of association of myosin heads with the thin filaments.d1,0 increased (38.9±0.1 versus 38.1±0.2nm, p 0.05) between the FE state and its reference state, and I1,1:I1,0 was increased (2.5±0.2 versus 1.3±0.1, p<0.05) in the FE state. These results suggest that the proportion of attached cross-bridges might be increased after active stretch and that this increase is not due to a decrease in lattice spacing but rather a change in cross-bridge kinetics.

Sarcomere Length Dependent Effects on Ca2+-Induced Troponin Regulation within Chemically Skinned Cardiac Muscle Fibers

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 sarcomere detects the SL changes and converts it into altered activation of thin filament. Among the myofilament proteins, troponin and myosin are two key components that are likely involved in the LDA. The two components are functionally linked through Ca2+ activation and cross-bridge feedback. Although the active cross-bridge feedback has been strongly implicated in LDA, there was no evidence linking sarcomere length changes and troponin regulation until our recent study showing that Ca2+-cTnC interaction in cardiac muscle can be modulated by sarcomere length through cross-bridge feedback [Biophysical Journal, 107(3), 682-93 (2014)]. In this study, we continue our efforts to understand the role of the C-domain of cTnI in LDA. Specifically, we used in situ time-resolved FRET measurements to determine how the switch region of cTnI is affected by Ca2+, sarcomere length, and cross-bridge in skinned cardiac muscle fibers. To monitor the Ca2+-induced structural transition of the switch region, skinned myocardial fibers were reconstituted with troponin complex containing FRET donor (AEDANS) modified cTnI(167C)AEDANS and acceptor (DDPM) modified cTnC(89C)DDPM. The measured FRET distance changes show that Ca2+, strong cross-bridges and sarcomere length all influence the structural transition of the switching region of cTnI within myocardial fibers. The results provide a mechanism by which sarcomere length can modulate Ca2+-troponin regulation via strong cross-bridge binding and suggest that the sarcomere length dependent cross-bridge effect plays an important role in the Frank-Starling law of the heart.

Cardiomyopathy Linked Mutations in Alpha Tropomyosin Influence Blocked State Stability but not Myosin Strong Binding

Coiled-coil tropomyosin binds super-helically along F-actin. Together with the troponin complex, tropomyosin inhibits myosin-binding. McKillop and Geeves (1993) introduced a three-state model for thin filament regulation with tropomyosin occupying three positions on the actin filament; blocked, closed and open. In the absence of myosin, tropomyosin primarily occupies the closed and blocked-states with the blocked-state being stabilized by Ca2+-free troponin. The probability of tropomyosin occupying any one of these two states is dependent on factors including cytosolic Ca2+-concentration (altering troponin conformation), mechanical strain, ionic strength, pH, and mutations that alter tropomyosin affinity for actin. Recently, we examined the impact of cardiomyopathy-linked tropomyosin mutations on the energy landscape of the tropomyosin/actin interaction. Our results demonstrated that mutations associated with hypertrophic cardiomyopathy showed reduced interaction-energy with actin resulting in a destabilization of the blocked-state. Using an in vitro motility assay here, we tested the ability of the mutant tropomyosins to regulate acto-myosin interactions, as well as the impact of strong binding cross-bridges on reconstituted thin filaments containing E40K and E62Q mutations in human alpha-tropomyosin (which lead to dilated (DCM) and hypertrophic (HCM) cardiomyopathies, respectively). Regulated thin filament velocity measurements showed an increase in low-Ca2+ contractility of E62Q reconstituted thin filaments when compared to wild-type and E40K reconstituted thin filaments. The ability of myosin to activate reconstituted mutant thin filaments at low-Ca2+ was not significantly different between the three tropomyosins, indicating that the enhanced motile fraction observed with E62Q is likely due to a destabilization of the blocked-state rather than alterations in myosin-dependent activation. The motility data is consistent with molecular dynamics simulations of the mutant tropomyosins in the presence of actin which show significant, yet subtle and complex alterations to the electrostatic interactions between tropomyosin and actin.

Bioelectrical effects of mechanical feedbacks in a strongly coupled cardiac electro-mechanical model

The aim of this work is to investigate by means of numerical simulations the effects of myocardial deformation due to muscle contraction on the bioelectrical activity of the cardiac tissue. The three-dimensional electro-mechanical model considered consists of the following four components: the quasi-static orthotropic finite elasticity equations for the deformation of the cardiac tissue; the active tension model for the intracellular calcium dynamics and cross-bridge binding; the orthotropic Bidomain model for the electrical current flow through the tissue; the membrane model of the cardiac myocyte, including stretch-activated currents (I SAC ). In order to properly take into account cardiac mechanical feedbacks, the electrical current flow is described in a strongly coupled framework by the Bidomain model on the deformed tissue. We then derive a novel formulation of the Bidomain model in the reference configuration, with complete mechanical feedbacks affecting not only the conductivity tensors but also a convective term depending on the velocity of the deformation. The numerical simulations are based on our finite element parallel solver, which employs both Multilevel Additive Schwarz preconditioners for the solution of linear systems arising from the discretization of the Bidomain equations and Newton–Krylov-Algebraic Multigrid methods for the solution of nonlinear systems arising from the discretization of the finite elasticity equations. The results have shown that: (i) the I SAC current prolongs action potential duration (APD) of about 10–15 ms; (ii) the inclusion into the model of both I SAC current and the convective term reduces the dispersion of repolarization of about 7% (from 139 to 129 ms) and increases the dispersion of APD about three times (from 13 to 45 ms). These effects indicate that mechanical feedbacks might influence arrhythmogenic mechanisms when combined with pathological substrates.

Quantifying inter-species differences in contractile function through biophysical modelling.

Animal models and measurements are frequently used to guide and evaluate clinical interventions. In this context, knowledge of inter-species differences in physiology is crucial for understanding the limitations and relevance of animal experimental assays for informing clinical applications. Extensive effort has been put into studying the structure and function of cardiac contractile proteins and how differences in these translate into the functional properties of muscles. However, integrating this knowledge into a quantitative description, formalising and highlighting inter-species differences both in the kinetics and in the regulation of physiological mechanisms, remains challenging. In this study we propose and apply a novel approach for the quantification of inter-species differences between mouse, rat and human. Assuming conservation of the fundamental physiological mechanisms underpinning contraction, biophysically based computational models are fitted to simulate experimentally recorded phenotypes from multiple species. The phenotypic differences between species are then succinctly quantified as differences in the biophysical model parameter values. This provides the potential of quantitatively establishing the human relevance of both animal-based experimental and computational models for application in a clinical context. Our results indicate that the parameters related to the sensitivity and cooperativity of calcium binding to troponin C and the activation and relaxation rates of tropomyosin/crossbridge binding kinetics differ most significantly between mouse, rat and human, while for example the reference tension, as expected, shows only minor differences between the species. Hence, while confirming expected inter-species differences in calcium sensitivity due to large differences in the observed calcium transients, our results also indicate more unexpected differences in the cooperativity mechanism. Specifically, the decrease in the unbinding rate of calcium to troponin C with increasing active tension was much lower for mouse than for rat and human. Our results also predicted crossbridge binding to be slowest in human and fastest in mouse.

A Coarse-Grained Model to Study Calcium Activation of the Cardiac Thin Filament

Familial hypertrophic cardiomyopathy (FHC) is one of the most common heart disease caused by genetic mutations. Cardiac muscle contraction and relaxation involve regulation of crossbridge binding to the cardiac thin filament, which regulates actomyosin interactions through calcium-dependent alterations in the dynamics of cardiac troponin (cTn) and tropomyosin (Tm). An atomistic model of cTn complex interacting with Tm has been studied by our group. A more realistic model requires the inclusion of the dynamics of actin filament, which is almost 6 times larger than cTn and Tm in terms of atom numbers, and extensive sampling of the model becomes very resource-demanding. By using physics-based protein united-residue force field, we introduce a coarse-grained model to study the calcium activation of the thin filament resulting from cTn's allosteric regulation of Tm dynamics on actin. In the corase-grained system, residues are represented by sidechain ellipsoids (SC) and peptide groups (p). Vectors between adjacent Cα-Cα and Cα-SC are chosen as variables, and the velocity Verlet algorithm is used to derive the equations of motion. The time scale is much longer than that of all-atom molecular dynamics simulation because of the reduction of the degrees of freedom. The coarse-grained model is a good template for studying cardiac thin filament mutations that cause FHC, and reduces the cost of computational resources.

Steady-State Predictions from a Compact Cooperative Kinetic Model of Cardiac Sarcomere Dynamics

A compact model of cardiac sarcomere dynamics is useful when developing integrated models of whole ventricular function. Kinetic models can be simplified by separating slower from faster processes. The representation of faster processes can then be reduced to their equivalent equilibrium relationships. Analysis of cooperativity within a cardiac sarcomere is also aided by dealing with equilibria, which can be analyzed using statistical thermodynamics. These principles guided the formulation of a compact kinetic model of cardiac sarcomere dynamics. In this scheme there were three slower processes: (1) reversible transitions between weak and strong crossbridges, (2) detachment of strong crossbridges, (3) dissociation of calcium from regulatory units (RUs) having a strong crossbridge. In the overall kinetic scheme, an individual RU could exist in 6 states: 3 states of crossbridge binding (none, weak, strong) interacting with 2 states of calcium binding (yes, no). Exchanges between the 4 states without strong crossbridges occurred by rapid equilibria. Thus, in the reduced model only 3 kinetic states remained: (1) strong crossbridges attached to RUs where calcium was bound, (2) strong crossbridges remaining attached even after calcium had dissociated, (3) the equilibrium mixture of the 4 remaining states. Since strong crossbridges appear to act independently, rate constants between these 3 states were considered independent of neighboring RUs. However, cooperativity between neighboring RUs exerted major impacts on the mixture of 4 states interacting rapidly as equilibria. Applying the linear Ising Model of statistical thermodynamics, the grand partition function characterizing interactions between neighboring RUs included activating effects from strong crossbridges as well as free calcium ions. This model predicted steady-state relationships between force and pCa (given as Hill plots) that reproduced the experimentally observed bend toward less cooperative slope near half activation.

Cardiac MyBP-C regulates the rate and force of contraction in mammalian myocardium.

Cardiac myosin-binding protein-C (cMyBP-C) is a thick filament-associated protein that seems to contribute to the regulation of cardiac contraction through interactions with either myosin or actin or both. Several studies over the past several years have suggested that the interactions of cardiac myosin-binding protein-C with its binding partners vary with its phosphorylation state, binding predominantly to myosin when dephosphorylated and to actin when it is phosphorylated by protein kinase A or other kinases. Here, we summarize evidence suggesting that phosphorylation of cardiac myosin binding protein-C is a key regulator of the kinetics and amplitude of cardiac contraction during β-adrenergic stimulation and increased stimulus frequency. We propose a model for these effects via a phosphorylation-dependent regulation of the kinetics and extent of cooperative recruitment of cross bridges to the thin filament: phosphorylation of cardiac myosin binding protein-C accelerates cross bridge binding to actin, thereby accelerating recruitment and increasing the amplitude of the cardiac twitch. In contrast, enhanced lusitropy as a result of phosphorylation seems to be caused by a direct effect of phosphorylation to accelerate cross-bridge detachment rate. Depression or elimination of one or both of these processes in a disease, such as end-stage heart failure, seems to contribute to the systolic and diastolic dysfunction that characterizes the disease.

2-Deoxy adenosine triphosphate improves contraction in human end-stage heart failure.

We are developing a novel treatment for heart failure by increasing myocardial 2 deoxy-ATP (dATP). Our studies in rodent models have shown that substitution of dATP for adenosine triphosphate (ATP) as the energy substrate in vitro or elevation of dATP in vivo increases myocardial contraction and that small increases in the native dATP pool of heart muscle are sufficient to improve cardiac function. Here we report, for the first time, the effect of dATP on human adult cardiac muscle contraction. We measured the contractile properties of chemically-demembranated multicellular ventricular wall preparations and isolated myofibrils from human subjects with end-stage heart failure. Isometric force was increased at both saturating and physiologic Ca(2+) concentrations with dATP compared to ATP. This resulted in an increase in the Ca(2+) sensitivity of force (pCa50) by 0.06 pCa units. The rate of force redevelopment (ktr) in demembranated wall muscle was also increased, as was the rate of contractile activation (kACT) in isolated myofibrils, indicating increased cross-bridge binding and cycling compared with ATP in failing human myocardium. These data suggest that dATP could increase dP/dT and end systolic pressure in failing human myocardium. Importantly, even though the magnitude and rate of force development were increased, there was no increase in the time to 50% and 90% myofibril relaxation. These data, along with our previous studies in rodent models, show the promise of elevating myocardial dATP to enhance contraction and restore cardiac pump function. These data also support further pre-clinical evaluation of this new approach for treating heart failure.