The HCM-Associated Cardiac Troponin T Mutation K280N Increases the Energetic Cost of Tension Generation in Human Cardiac Myofibrils

Abstract

We have used site-directed spin labeling and pulsed dipolar electron-electron paramagnetic resonance (DEER) to resolve the structure and dynamics of flexible and disordered regions of myosin binding protein-C (MyBP-C)'s cardiac isoform, with implications for the pathophysiology of hypertrophic cardiomyopathy (HCM). N-terminal domains of cMyBP-C contain binding domains for several interaction partners in the myofilament, including myosin heavy chain subfragment 2 (S2) and actin. We engineered pairs of labeling sites in protein fragments of mouse cMyBP-C to measure with high resolution distance and disorder between (1) domains C0 and C1, flanking the flexible Pro/Ala-rich linker, and between (2) domains C1 and C2, flanking the partially disordered phosphorylation motif, using DEER. Changes in distance and disorder were assessed for double-Cys mutant cMyBP-C's free in solution and when bound to myosin S2 or actin, with or without cMyBP-C phosphorylation by protein kinase A (PKA). Understanding conformational transitions in the flexible and dynamic portions of cMyBP-C upon actin-myosin binding and phosphorylation provide new molecular insight into defining its modulatory role in muscle force development.

Abnormal contractility in human heart myofibrils from patients with dilated cardiomyopathy due to mutations in TTN and contractile protein genes

“…In contrast to the studies on human heart tissue from HCM patients 21 , we found the maximum isometric force was not diminished, and length dependent activation of maximal force was not altered in any of the DCM mutants. This is consistent with our detailed measurements using myofibrils from the ACTC E361G DCM transgenic mouse 11 , 21 – 23 .…”

Mutations in troponin T associated with Hypertrophic Cardiomyopathy increase Ca2+-sensitivity and suppress the modulation of Ca2+-sensitivity by troponin I phosphorylation

“…In contrast to measurements of Ca 2+ -sensitivity, the effect of the mutation on the kinetics of myofibrillar force production and relaxation at saturating Ca 2+ concentrations showed that exchange of recombinant human cardiac TnT K280N into donor and also of wild-type troponin into TnT K280N patient myofibrils gave fully reversible effects [33] . Since IVMA measures unloaded movement at 29 °C, whilst studies on myofibrils or permeabilised cells measure isometric force only at saturating Ca 2+ at 15 °C, the studies are not directly comparable as we discussed previously [17] and therefore not contradictory.…”

“…This sample enabled us to study troponin with an HCM related mutation in TnT in the context of the patient's heart. Contractility in the same sample has also been studied in skinned muscle fibres and in single myofibrils [33] . Both the isolated troponin and the skinned muscle fibres indicated that myofilament Ca 2+ -sensitivity was not very different from donor heart controls: the two were indistinguishable in IVMA whilst TnT K280N patient skinned muscle had a 20% higher Ca 2+ -sensitivity in skinned fibres.…”