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

BACKGROUND

Impaired left ventricular relaxation, high filling pressures, and dysregulation of Ca 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 , and via 2D echocardiography, western blotting, 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 D ) was sufficient to recover diastolic function to Non-Tg levels only for R92L-cTnT. Mutation-specific changes in Ca dissociation rates associated with R92L-cTnT reconstituted with cTnI-D D 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 β-adrenergic responsiveness and identifying a potential molecular target for therapeutic intervention.