Furthering the development of in vitro models in cardiovascular research: focus on a TITIN truncating variant associated with atrial fibrillation

Cumberland, Max (2024). Furthering the development of in vitro models in cardiovascular research: focus on a TITIN truncating variant associated with atrial fibrillation. University of Birmingham. Ph.D.

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Abstract

Background: Induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) have revolutionised cardiac disease modelling over the past decade and are causing an inexorable shift towards the use of in vitro models. However, the utility of iPSC-CMs is still limited by the immaturity of the cells derived and their inability to recapitulate the complexities of cardiac disease. Co-culture 3D cardiac models confer tangible improvements in cell maturity and enable researchers to investigate the heterocellular cell to cell and cell to matrix interactions integral to the pathophysiology of heart disease.

Atrial fibrillation (AF) is a supraventricular tachyarrhythmia characterised by the rapid and irregular beating of the upper chambers of the heart. Dilated cardiomyopathy (DCM) is a type of heart disease that causes inefficient pumping function, which leads to heart failure. Pathogenic variants that cause a truncation in the gigantic protein titin (TTNtv) are a genetic risk factor for the development of AF and DCM. The mechanism by which TTNtv can cause AF is currently obscure. The TTNtv, c.59926 + 1G > A, was previously identified as being causative in the development of DCM and AF.

Aim: Generate and validate atrial iPSC-CMs for compartment specific markers (Chapter 2). Develop an atrial 3D co-culture cardiac model using iPSC derived cardiomyocytes and cardiac fibroblasts (iPSC-CFs) (Chapter 3). Model the effect an AF predisposing TTNtv has on electrical and structural remodelling in vitro (Chapter 4). Investigate the utility of iPSCCFs in exploring the pathophysiology of cardiac fibrosis (Chapter 5).

Methods: Atrial iPSC-CMs were generated and assessed for transcriptional, structural, and electrophysiological markers of compartment specificity. iPSC-CFs were differentiated, validated and incorporated into a 3D co-culture cardiac model. The c.59926 + 1G > A variant was engineered into iPSCs using CRISPR-Cas9 gene editing. The effect the variant had on electrical and structural remodelling was assessed in vitro using patch clamp analysis and molecular biology techniques.

Results: The differentiated atrial iPSC-CMs demonstrated compartment specific transcriptional, structural and electrophysiological characteristics (Chapter 2). Addition of iPSC-CFs into a 3D co-culture model improved tissue compaction, contractile function and viability (Chapter 3). Consistent with previous in vivo studies, the TTNtv modelled increased the propensity for atrial fibrosis. Disruption to cholinergic stimulation in TTNtv atrial cardiomyocytes was observed in vitro and proved pro-arrhythmic when modelled in silico (Chapter 4). iPSC-CFs offered a more practical model than primary cardiac fibroblasts in which to assess cardiac fibrosis (Chapter 5).

Conclusions: This thesis evidences the utility of iPSC-CM/iPSC-CF 3D cardiac models in exploring the complexities of heart disease. For the first time, a TTNtv is shown to increase propensity for atrial fibrosis in vitro. Supporting previous evidence that TTNtv cause atrial remodelling and acting as valid mechanism for AF. Dysregulated cholinergic stimulation of cardiomyocytes is caused by TTNtv and represents a novel pathomechanism for arrhythmia.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):