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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. 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. 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. 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. Through Brownian dynamics simulation and computational structural analysis, we found that deoxy-hydrolysis products [2-deoxy-adenosine 5'-diphosphate (dADP) and inorganic phosphate (Pi)] bound to prepowerstroke myosin induce an allosteric restructuring of the actin-binding surface on myosin to increase the rate of cross-bridge formation. We then show experimentally that this predicted effect translates into increased electrostatic interactions between actin and cardiac myosin in vitro. 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. 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.

Hallmark features of systolic heart failure are reduced contractility and impaired metabolic flexibility of the myocardium. Cardiomyocytes (CMs) with elevated deoxy ATP (dATP) via overexpression of ribonucleotide reductase (RNR) enzyme robustly improve contractility. However, the effect of dATP elevation on cardiac metabolism is unknown. Here, we developed proteolysis-resistant versions of RNR and demonstrate that elevation of dATP/ATP to ∼1% in CMs in a transgenic mouse (TgRRB) resulted in robust improvement of cardiac function. Pharmacological approaches showed that CMs with elevated dATP have greater basal respiratory rates by shifting myosin states to more active forms, independent of its isoform, in relaxed CMs. Targeted metabolomic profiling revealed a significant reprogramming towards oxidative phosphorylation in TgRRB-CMs. Higher cristae density and activity in the mitochondria of TgRRB-CMs improved respiratory capacity. Our results revealed a critical property of dATP to modulate myosin states to enhance contractility and induce metabolic flexibility to support improved function in CMs. [Display omitted] • Ubiquitylation-resistant variant RRB in a transgenic mice model (TgRRB) elevates dATP in the heart and improves function. • TgRRB-CMs show greater basal oxygen consumption due to changes in myosin state by dATP. • TgRRB-CMs respond to elevated function with higher pools of oxidative metabolites, elevated OXPHOS, FAO, and energy reserve. • Long-term mitochondrial remodeling may occur to accommodate for the higher energy demands of the high functioning TgRRB-CMs. [ABSTRACT FROM AUTHOR]

Key points: Skeletal muscle relaxation has been primarily studied by assessing the kinetics of force decay. Little is known about the resultant dynamics of structural changes in myosin heads during relaxation.The naturally occurring nucleotide 2‐deoxy‐ATP (dATP) is a myosin activator that enhances cross‐bridge binding and kinetics.X‐ray diffraction data indicate that with elevated dATP, myosin heads were extended closer to actin in relaxed muscle and myosin heads return to an ordered, resting state after contraction more quickly.Molecular dynamics simulations of post‐powerstroke myosin suggest that dATP induces structural changes in myosin heads that increase the surface area of the actin‐binding regions promoting myosin interaction with actin, which could explain the observed delays in the onset of relaxation.This study of the dATP‐induced changes in myosin may be instructive for determining the structural changes desired for other potential myosin‐targeted molecular compounds to treat muscle diseases. Here we used time‐resolved small‐angle X‐ray diffraction coupled with force measurements to study the structural changes in FVB mouse skeletal muscle sarcomeres during relaxation after tetanus contraction. To estimate the rate of myosin deactivation, we followed the rate of the intensity recovery of the first‐order myosin layer line (MLL1) and restoration of the resting spacing of the third and sixth order of meridional reflection (SM3 and SM6) following tetanic contraction. A transgenic mouse model with elevated skeletal muscle 2‐deoxy‐ATP (dATP) was used to study how myosin activators may affect soleus muscle relaxation. X‐ray diffraction evidence indicates that with elevated dATP, myosin heads were extended closer to actin in resting muscle. Following contraction, there is a slight but significant delay in the decay of force relative to WT muscle while the return of myosin heads to an ordered resting state was initially slower, then became more rapid than in WT muscle. Molecular dynamics simulations of post‐powerstroke myosin suggest that dATP induces structural changes in myosin that increase the surface area of the actin‐binding regions, promoting myosin interaction with actin. With dATP, myosin heads may remain in an activated state near the thin filaments following relaxation, accounting for the delay in force decay and the initial delay in recovery of resting head configuration, and this could facilitate subsequent contractions. Key points: Skeletal muscle relaxation has been primarily studied by assessing the kinetics of force decay. Little is known about the resultant dynamics of structural changes in myosin heads during relaxation.The naturally occurring nucleotide 2‐deoxy‐ATP (dATP) is a myosin activator that enhances cross‐bridge binding and kinetics.X‐ray diffraction data indicate that with elevated dATP, myosin heads were extended closer to actin in relaxed muscle and myosin heads return to an ordered, resting state after contraction more quickly.Molecular dynamics simulations of post‐powerstroke myosin suggest that dATP induces structural changes in myosin heads that increase the surface area of the actin‐binding regions promoting myosin interaction with actin, which could explain the observed delays in the onset of relaxation.This study of the dATP‐induced changes in myosin may be instructive for determining the structural changes desired for other potential myosin‐targeted molecular compounds to treat muscle diseases. [ABSTRACT FROM AUTHOR]