Arg92Leu-cTnT Alters the cTnC-cTnI Interface Disrupting PKA-Mediated Relaxation.

Abstract

BACKGROUND Impaired left ventricular relaxation, high filling pressures, and dysregulation of Ca2+ 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 the potential involvement of myofilament regulators in relaxation. 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 cTnC (cardiac troponin C)-cTnI (cardiac troponin I) interface can account for the development of early-onset diastolic dysfunction via decreased PKA accessibility to cTnI. METHODS HCM mutations R92L-cTnT (cardiac troponin T; Arg92Leu) and Δ160E-cTnT (Glu160 deletion) were studied in vivo, in vitro, and in silico via 2-dimensional echocardiography, Western blotting, ex vivo hemodynamics, stopped-flow kinetics, time-resolved fluorescence resonance energy transfer, and molecular dynamics simulations. RESULTS The HCM-causative mutations R92L-cTnT and Δ160E-cTnT result in different time-of-onset diastolic dysfunction. R92L-cTnT demonstrated early-onset diastolic dysfunction accompanied by a localized decrease in phosphorylation of cTnI. Constitutive phosphorylation of cTnI (cTnI-D23D24) was sufficient to recover diastolic function to non-Tg levels only for R92L-cTnT. Mutation-specific changes in Ca2+ dissociation rates associated with R92L-cTnT reconstituted with cTnI-D23D24 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 time-resolved fluorescence resonance energy transfer 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 time-resolved fluorescence resonance energy transfer distances as constraints into our atomistic model identified additional electrostatic interactions at the consensus sequence. CONCLUSIONS These data show that the early diastolic dysfunction observed in a subset of HCM is attributable to allosterically mediated structural changes at the cTnC-cTnI interface that impair accessibility of PKA, thereby blunting β-adrenergic responsiveness and identifying a potential molecular target for therapeutic intervention.