The HCM – Linked Mutation Arg92Leu in TNNT2 Allosterically Alters the cTnC – cTnI Interface and Disrupts the PKA-mediated Regulation of Myofilament Relaxation

Abstract

ABSTRACTBackgroundImpaired 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 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.MethodsHCM mutations R92L-cTnT (Arg92Leu) and Δ160E-cTnT (Glu160 deletion) were studiedin vivo,in vitro,andin silicovia 2D echocardiography, western blotting,ex vivohemodynamics, stopped-flow kinetics, time resolved fluorescence resonance energy transfer (TR-FRET), and molecular dynamics simulations.ResultsThe 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-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-D23D24led 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.ConclusionThese 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 β-adrenergic responsiveness and identifying a potential molecular target for therapeutic intervention.