Taleb, Yaqob Samir A;
(2023)
Electrophysiological and cellular analysis of
filamin-C mutations causing cardiomyopathy
using human iPSC-derived cardiomyocytes.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Background: Arrhythmogenic Cardiomyopathy (AC) is a genetic cardiac disease resulting from different mutations within proteins constituting the intercalated disc, including desmosomal and nondesmosomal proteins. Recent studies have revealed that mutations in filamin-C (FLNC) may lead to AC. The arrhythmogenesis and electrophysiological effects of FLNC-related AC are incompletely understood. Therefore, the aim of this study is to assess the potential electrophysiological consequences of FLNC loss as occurs in AC in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs). Specifically, I aimed to characterise abnormal electrical activity and the expression and function of key proteins in cardiac electrical activity such as gap junction protein connexin 43 (Cx43).// Methods: hiPSC-CMs were differentiated and observed by immunofluorescence microscopy. Small interfering RNA (siRNA) transfection was utilised to knockdown the expression of FLNC in hiPSC-CMs. Protein analysis was performed using western blotting to confirm the knockdown efficiency. Electrophysiological properties were recorded using a multielectrode array and manual patch clamping. Optical recording of membrane potential and calcium activity from hiPSC-CMs were also carried out using parameter sensitive dyes.// Results: Silencing of FLNC led to markedly decreased immunofluorescence signals of FLNC, Cx43, desmoplakin, and junctional plakoglobin. No significant reductions were noted in the immunofluorescence signals of voltage-gated sodium channel (Nav1.5) and plakophilin-2 compared with control hiPSC-CMs. Western blotting showed the reduction of FLNC and Cx43 expression following silencing of FLNC. Knockdown of FLNC resulted in disturbances to the recorded action and field potential signals of hiPSC-CMs and arrhythmic likeevents. Transfected hiPSC-CMs with siRNA-FLNC were associated with prolongation of calcium transient durations, optical action potential duration, and action potentials measured with patch clamping.// Conclusion: The current findings indicated that loss of FLNC resulted in a complex arrhythmogenic phenotype in hiPSC-CMs
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Electrophysiological and cellular analysis of filamin-C mutations causing cardiomyopathy using human iPSC-derived cardiomyocytes |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2023. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > Institute of Cardiovascular Science |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10177464 |
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