Cryo-EM Studies of the Heteromultimeric Cardiac Thin Filament

Abstract

The thin filament is a crucial component of cardiac muscle. The heteromultimeric protein regulates contraction through its constituent proteins: actin, tropomyosin, troponin C, troponin I, and troponin T. Mutations in all these proteins can cause familial hypertrophic cardiomyopathy, the most common form of inherited heart disease. Recent advancements in cryo-electron microscopy (cryo-EM) have revealed new fundamental structural details and regulatory mechanisms, although important regions remain unmodelled.
I have used zebrafish (danio rerio) as a model system to study the structure of the native cardiac thin filament. Zebrafish are a widely used animal model for cardiovascular research, benefiting from conserved regulatory and gene pathways, high fecundity, rapid development, and high genetic tractability.
Using single particle cryo-EM I have demonstrated the structural suitability of zebrafish for the study of human cardiovascular disease. The first 3D reconstruction of the zebrafish native thin filament shows the same organisational structure as recombinant human cryo-EM data.
Resolution limitations were encountered due to the loss of regulatory proteins during sample preparation. This was partially mitigated by chemical fixation techniques improving protein retention and image processing strategies were trialled to recover fully decorated thin filaments. The dominant population in this fixed data was actin decorated with the regulatory protein tropomyosin, for which high-resolution reconstructions were achieved.
Large populations of bare F-actin were present in native thin filament preparations. A high-resolution imaging workflow was pursued and a near-atomic 3.16 Å resolution structure of zebrafish actin was determined. Molecular modelling was performed allowing in-depth analysis of the nucleotide state, the nucleotide-associated divalent cation and D-loop orientation.
Using the developed methods and techniques outlined in this thesis, further work combined with genetic manipulation will help us to investigate physiological and pathophysiological function of the thin filament potentially leading the way for the design of new targeted therapies.

Date of Award	1 Oct 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Danielle M Paul (Supervisor) & Massimo Caputo (Supervisor)