Characterising the palmitoylation and SUMOylation of cardiac myosin binding protein-C in cardiac health and disease

Main, Alice (2022) Characterising the palmitoylation and SUMOylation of cardiac myosin binding protein-C in cardiac health and disease. PhD thesis, University of Glasgow.

Abstract

Cardiac myosin binding protein-C (cMyBP-C) is a 12-domain sarcomeric accessory protein that transiently interact with actin, tropomyosin and myosin and regulates the activity of the myofilament to maintain systolic and diastolic function. cMyBPC is influenced by an increasing list of post-translational modifications (PTMs), including phosphorylation, which occurs predominantly in the N-terminal regions and regulates myofilament force and calcium sensitivity. Whilst the central domains have remained lesser studied, evidence suggests they are may promote different conformations of cMyBP-C, influence myosin binding and are a hot-spot for PTMs. This includes the cysteine modification S-glutathionylation, an increase of which impairs cMyBP-C phosphorylation and increases myofilament calcium sensitivity. In this study, the cysteine modification palmitoylation was investigated, which has not been widely reported for myofilament proteins. Acyl resin assisted capture (Acyl-RAC) was used to purify palmitoylated proteins from cardiac tissue and revealed that actin, myosin and cMyBP-C undergo palmitoylation. Upon investigation of different anatomical regions, cMyBP-C palmitoylation may be highest in the left ventricle and appears reduced when these primary cardiomyocytes are cultured. Furthermore, the palmitoylated form of cMyBP-C may be more resistant to salt extraction from the myofilament lattice. In cardiac pathologies, palmitoylation was reduced in the left ventricle of a rabbit model of heart failure (HF) but increased in ischaemic human HF samples. Site directed mutagenesis revealed C623 and C651, in the C4 and C5 domains respectively, to be candidate palmitoylation sites, which have previously been identified to be modified by S-glutathionylation. Isolated myofilaments treated with palmitoyl CoA, which spontaneously attaches to palmitoylated cysteines, showed significantly increased levels of cMyBP-C palmitoylation and reduced calcium sensitivity of force. Whether this is attributed solely to cMyBP-C palmitoylation remains to be determined, nevertheless this study provides novel evidence that palmitoylation is an important regulatory modification for myofilament function. Aside from palmitoylation, preliminary data suggests cMyBP-C also undergoes SUMOylation. This was investigated using a cMyBP-C-UBC9 fusion construct (WT) co-expressed with eGFP-SUMO1, which shows a SUMOylated band shift, and a catalytically inactive mutant (C93A) which cannot be SUMOylated. Purification of the SUMOylated cMyBP-C-UBC9 fusion for mass spectrometry and in silico analysis identified several candidate SUMOylation sites, however individual mutation did not result in the loss of the SUMOylated band. Reduced phosphorylation of SUMOylated form of cMyBP-C-UBC9 was observed in HEK293 cells and in virally infected neonatal ventricular cardiomyocytes treated with isoprenaline, which also show a blunted lusitropic response to isoprenaline. This may indicate that SUMOylation of cMyBP-C can regulate cardiac contractility, however experimental limitations, including lack of in-situ evidence that cMyBP-C is SUMOylated, limit the conclusions that can be drawn from this study. Given the evidence presented here that cMyBP-C palmitoylation is altered in HF, the palmitoylation of other key cardiac substrates was investigated and were found to be altered in animal models and human HF patients in a similar manner. Animal models of cardiac hypertrophy and HF were generally associated with a loss of palmitoylation, whilst human HF showed increased palmitoylation of substrates including NCX1 and Na+/K+ ATPase. As NCX1 is a reported substrate, expression and palmitoylation of DHHC5 was evaluated in these samples. Cardiac hypertrophy was associated with an increase in DHHC5 expression as early as 3- days post injury, however HF development was associated with unchanged or reduced levels of DHHC5. Previous work suggests DHHC5 overexpression may not directly impact protein palmitoylation or cardiomyocyte function, therefore DHHC5 palmitoylation was evaluated to investigate whether its activity may be changed. Interestingly, DHHC5 palmitoylation followed a similar pattern in disease to NCX1. This may indicate that there are upstream factors such as fatty acid availability that influence the palmitoylation of all substrates together. This study provides an insight into changes of palmitoylation in cardiac disease, although given that changes in singly palmitoylated proteins are more easily detected by Acyl-RAC, further characterisation using additional methods is required.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Supervisor's Name: Fuller, Prof. Will, Gadegaard, Prof. Nikolaj and Baillie, Prof. George
• Date of Award: 2022
• Depositing User: Theses Team
• Unique ID: glathesis:2022-83125
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Sep 2022 10:52
• Last Modified: 14 Sep 2022 11:12
• Thesis DOI: 10.5525/gla.thesis.83125
• URI: https://theses.gla.ac.uk/id/eprint/83125

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