ABSTRACT
Background: Impaired left ventricular relaxation, high filling pressures, and dysregulation of Ca 2+ homeostasis are common findings contributing to diastolic dysfunction in hypertrophic cardiomyopathy (HCM). Studies have shown that impaired relaxation is an early observation in the sarcomere-gene-positive preclinical HCM cohort which suggests potential involvement of myofilament regulators of relaxation. Yet, a molecular level understanding of mechanism(s) at the level of the myofilament is lacking. We hypothesized that mutation-specific, allosterically mediated, changes to the cardiac troponin C-cardiac troponin I (cTnC-cTnI) interface can account for the development of early-onset diastolic dysfunction via decreased PKA accessibility to cTnI. Methods: HCM mutations R92L-cTnT (Arg92Leu) and Δ160E-cTnT (Glu160 deletion) were studied in vivo , in vitro, and in silico via 2D echocardiography, western blotting, ex vivo hemodynamics, stopped-flow kinetics, time resolved fluorescence resonance energy transfer (TR-FRET), and molecular dynamics simulations. Results: The HCM-causative mutations R92L-cTnT and Δ160E-cTnT result in different time-of-onset of diastolic dysfunction. R92L-cTnT demonstrated early-onset diastolic dysfunction accompanied by a localized decrease in phosphorylation of cTnI. Constitutive phosphorylation of cTnI (cTnI-D 23 D 24 ) was sufficient to recover diastolic function to Non-Tg levels only for R92L-cTnT. Mutation-specific changes in Ca 2+ dissociation rates associated with R92L-cTnT reconstituted with cTnI-D 23 D 24 led us to investigate potential involvement of structural changes in the cTnC-cTnI interface as an explanation for these observations. We probed the interface via TR-FRET revealing a repositioning of the N-terminus of cTnI, closer to cTnC, and concomitant decreases in distance distributions at sites flanking the PKA consensus sequence. Implementing TR-FRET distances as constraints into our atomistic model identified additional electrostatic interactions at the consensus sequence. Conclusion: These data indicate that the early diastolic dysfunction observed in a subset of HCM is likely attributable to structural changes at the cTnC-cTnI interface that impair accessibility of PKA thereby blunting ß-adrenergic responsiveness and identifying a potential molecular target for therapeutic intervention.
ABSTRACT
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.
