BD simulations of prepowerstroke myosin binding to an actin dimer with either ADP or dADP suggest that dADP-myosin has an increased number of polar residue interactions at the actin–myosin interface compared with ADP-myosin, which is predicted to increase the association kinetics. Specifically, atomistic structural models of prepowerstroke myosin-ADP, myosin-dADP, and actin were used to investigate how each nucleotide affects the steric orientation of the actin–myosin-binding surface and the energetics associated with the actin–myosin complex.

As such, we developed an approach to investigate the mechanisms of action of a specific inotropic agent (dATP) with potential therapeutic applications in cardiac muscle that incorporate atomically detailed analysis of molecular structure, Brownian dynamics (BD) simulations of actin–myosin-binding kinetics, and small-angle X-ray diffraction analysis of sarcomere structure. However, assessing how small changes in myosin structure translate to large changes in sarcomere dynamics and contractility is a challenge that requires a combination of multiple techniques that span multiple spatiotemporal scales. This led us to hypothesize that dATP increases the electrostatic interactions between cardiac myosin and actin, which may alter the conformation of myosin in the sarcomere to prime myosin for binding to actin and increase cross-bridge formation during activation. A recent computational study using molecular dynamics simulations suggests that dATP behaves as an allosteric effector of prepowerstroke myosin, such that when 2-deoxy-adenosine 5′-diphosphate and inorganic phosphate (dADP.Pi) are in the nucleotide binding pocket, local structural changes lead to exposure of more positively charged residues on the actin-binding surface of myosin compared withADP.Pi ( 8). Chemomechanical studies suggest that the dATP-mediated force augmentation occurs by increasing cross-bridge binding and cycling rates ( 4– 7), but a precise structural explanation for this is lacking. Moreover, we have shown that dATP increases contractility in rat myocardium ( 1), improves left ventricular function in a porcine model of myocardial infarction ( 2), and increases contractility of ventricular muscle from patients in human end-stage HF ( 3).

We have previously demonstrated that dATP can be used by cardiac myosin in place of ATP as the substrate that fuels actin–myosin cross-bridge cycling ( 1). One is the naturally occurring ATP analog 2-deoxy-ATP (denoted dATP). Accordingly, there are several myosin-specific small-molecule compounds currently being developed and tested to treat HF. The dATP-mediated structural alterations in myosin reported here may provide insight into an improved criterion for the design or selection of small molecules to be developed as therapeutic agents to treat systolic dysfunction.Ĭardiac muscle contraction is generated at the molecular level by cyclical, adenosine 5′-triphosphate (ATP)–driven actin–myosin interactions, making myosin an appealing therapeutic target for treating ventricular systolic dysfunction. Finally, using small-angle X-ray diffraction analysis of sarcomere structure, we demonstrate that the proposed increased electrostatic affinity of myosin for actin causes a disruption of the resting conformation of myosin motors, resulting in their repositioning toward the thin filament before activation.

We then show experimentally that this predicted effect translates into increased electrostatic interactions between actin and cardiac myosin in vitro. Through Brownian dynamics simulation and computational structural analysis, we found that deoxy-hydrolysis products bound to prepowerstroke myosin induce an allosteric restructuring of the actin-binding surface on myosin to increase the rate of cross-bridge formation. Exploiting dATP as a molecular probe, we assess how small changes in myosin structure translate to electrostatic-based changes in sarcomere function to augment contractility in cardiac muscle. In this work, we combine multiple techniques to provide structural and functional information at the angstrom-nanometer and millisecond time scales, demonstrating the ability to make both structural measurements and quantitative kinetic estimates of weak actin–myosin interactions that underpin sarcomere dynamics. We and others have previously shown that dATP increases contractile force in normal hearts and models of depressed systolic function, but the structural basis of these effects has remained unresolved. The naturally occurring nucleotide 2-deoxy-adenosine 5′-triphosphate (dATP) can be used by cardiac muscle as an alternative energy substrate for myosin chemomechanical activity.

Deoxy i ching