Abstract
The coordinated activation and de-activation of both the actin-containing thin and myosin-containing thick filaments of the sarcomere, the basic contractile unit of the heart, is fundamental for normal heart muscle function. Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated regulatory protein localised to the inner two thirds of the half-sarcomere A-band, called the ‘C-zone’, via interactions of its C-terminal domains with titin and myosin tail domains. Recent in vitro experiments have highlighted the functional significance of the N-terminal region of cMyBP-C for heart muscle contraction, reporting regulatory interactions with both thick and thin filaments. Moreover, pathogenic variants in the gene encoding for cMyBP-C (MYBPC3) are the second most common cause of heritable hypertrophic cardiomyopathy, further underlining its functional significance.To better understand the interactions of the regulatory N-terminal domains of cMyBP-C in its native sarcomere environment, in situ FRET-FLIM binding assays were developed in order to determine the spatial relationship between the N-terminus of full-length cMyBP-C and the thick and thin filament in demembranated cardiomyocytes in the well characterised steady states of relaxation and rigor.
Control studies were performed using established biochemical and biophysical methods to determine the effects of introducing the fluorophores for FRET into sarcomeric proteins. The mTFP- and mVenus- labelled cMyBP-C, cRLC and cTnT had the same sarcomeric location as the endogenous proteins, to the C-zone, thick filament A-band and thin filament, respectively, when expressed in intact or chemically skinned neonatal rat cardiomyocytes (NRCs). In vitro studies showed that ligation of a genetically-encoded fluorophore to the N-terminus of cMyBP-C had no or little effect on its binding to thick and thin filament proteins, on the ATPase activity of isolated cardiac myosin in the presence of native thin filaments, or on PKA-mediated phosphorylation of the three key sites in the m-motif of cMyBP-C.
After optimisation of the FRET-FLIM workflow, FRET was detected between N-terminally labelled mTFP-cMyBP-C as the donor and phalloidin-iFluor 514 labelling the actin filaments as the acceptor in skinned NRCs in both relaxing and rigor conditions. The measured FRET efficiency was intermediate between those observed when the donor was on the N-termini of cRLC and cTnT. A broad distribution of FRET efficiencies was observed between cMyBP-C and phalloidin, suggesting a broad distribution of distances between the N-terminal domains of cMyBP-C and the thin filament, particularly in relaxing conditions. These results are consistent with the co-existence of multiple populations of cMyBP-C, with a large proportion of their N-terminal domains binding to the thin filament and others binding to the thick filament, supporting the hypothesis that the dynamic interchange between populations mediates inter- filament signalling in the regulation of contractility.
| Date of Award | 1 Jan 2022 |
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| Original language | English |
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| Supervisor | Malcolm Irving (Supervisor) & Thomas Kampourakis (Supervisor) |