Linking molecular targets to hypertrophic cardiomyopathy using genome-edited human induced pluripotent stem cell cardiomyocytes

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Bodbin, S.E. (2022) Linking molecular targets to hypertrophic cardiomyopathy using genome-edited human induced pluripotent stem cell cardiomyocytes. PhD thesis, University of Nottingham.

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Abstract

Hypertrophic cardiomyopathy (HCM) is the most common cardiomyopathy, estimated to affect 1:250 people, resulting in impaired cardiac function due to thickening of the left ventricle. Once disregarded as genomic junk, long non-coding ribonucleic acids (lncRNA) have emerged as key regulators of human disease, including cardiovascular disorders. Work prior to this thesis developed an hiPSC- CM model of HCM, introducing the pathological R453C mutation into the MYH7 sarcomeric gene. RNA sequencing on the lines of the of model uncovered four lncRNAs that may be involved in the progression or prevention of HCM.

Current transfection methods pose a barrier to the full potential hiPSC-CMs offer. In absence of a suitable transfection method and the criticality of efficient transfection prior to the phenotyping assays, a new method was optimised specifically for this thesis. Using a cationic reagent and through systematic optimisation of several conditions, a high transfection efficiency without compromising cardiomyocyte purity and with little effect on cell viability was achieved.

Expression modulation systems were harnessed for the probing of the phenotypic effects of the lncRNA targets on HCM, with the aim of providing a new avenue for pharmacological therapeutics. CRISPR/Cas9 technology was implemented for the creation of stable knock outs of the lncRNAs in each of the isogenic lines. One of the lncRNAs, MINCR, revealed novel characteristics upon knock out in hiPSCs.

A monolayer cardiac differentiation protocol was applied to the three lines of the HCM model, after which hiPSC-CMs were transfected with overexpression vectors in addition to small interfering RNAs (siRNA) to achieve knock down. Functional phenotyping was performed on hiPSC-CMs with up or downregulated lncRNA target expression. Assessing brain natriuretic protein (BNP) as a marker of hypertrophy, suggested that the lncRNAs in question act as gene modifiers, promoting or preventing HCM depending on the MYH7 cell line. Probing the lncRNA involvement in mitochondrial respiration did not reveal any immediate lncRNA involvement thus far. Finally, assessing the switch between the two

isoforms of myosin heavy chain, a hallmark of HCM, revealed the potential involvement of lncRNAs in regulating their expression, with further optimisation of the methods required.

The work in this thesis optimised a transfection protocol for successful hiPSC-CM transfection which is critical for the investigation of disease mechanisms in cardiomyocytes. In addition, results from phenotyping lncRNA expression modulated hiPSC-CMs of the HCM model highlights the role of the lncRNA targets in acting as gene modifiers that impact HCM in a cardioprotective or disease- causing manner.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Denning, C.
Keywords: iPSC-CM, stem cell biology, disease modelling, CRISPR
Subjects: W Medicine and related subjects (NLM Classification) > WG Cardiocascular system
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID: 71672
Depositing User: Bodbin, Sara
Date Deposited: 15 Dec 2022 04:40
Last Modified: 15 Dec 2022 04:40
URI: https://eprints.nottingham.ac.uk/id/eprint/71672

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