Use of induced pluripotent stem cell derived cardiomyocytes to test the consequences of genetic variants in atrial and ventricular arrhythmias

Why did we fund this project?

This award aims to use induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) to investigate inherited cardiac arrhythmias, replacing the use of transgenic mouse models.

Inherited genetic mutations are the most common cause of sudden cardiac death as they increase predisposition to abnormal heart rhythm. These include titin truncation variants (TTNtv), which can impact on the heart in a number of ways including changes in stiffness and elasticity. Animal models, typically transgenic mice, can be genetically modified to possess the same mutations. These require large breeding programmes to generate the mutations of interest. Electrocardiograms or implanted telemeters are then used to assess the impact of the mutation on the cardiovascular system. Human iPSC-CMs present an alternative model to study inherited heart conditions and have a number of scientific advantages over the use of mouse models, both in vivo and ex vivo, for example, mice have different heart rates and ion channels to humans.

Optimised genome editing protocols will be used to introduce TTNtv mutations into iPSCs. The student will combine this technology with 3D culturing methods to produce engineered heart tissue, which should result in iPSC-CMs with more mature characteristics that are functionally similar to adult cardiomyocytes and a better model of disease. The student will also develop skills in differentiation protocols, optical mapping, and in silico modelling of electric cardiac disturbances.

The project is additionally supported by the expertise and input of collaborators, Professor Chris Denning, University of Nottingham, Dr Ben Davies, University of Oxford and Dr Andrew Holmes, University of Birmingham.

This Studentship was co-awarded with the British Heart Foundation (BHF).

Inherited cardiac conditions are caused by genetic mutations in cardiac genes. They are the most common cause of sudden cardiac death in the young through ventricular arrhythmias and also contribute substantially to heart failure, putting a major burden on health care systems. To develop specific therapies, it is necessary to understand the consequences of the genetic mutations at the molecular level. Model systems, such as cells, organoids and whole organisms are used to elucidate disease pathways. The animal models among them have a high ethical burden, therefore there is a need to replace them by cellular models.

Induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) have emerged as a novel model system to study inherited heart conditions and thereby provide an opportunity to replace animal models, especially mice, to study these genetic diseases. The advantage of the iPSC-CM system is their human physiology (e.g. beating rates and ion channel setups) and that genetic mutations can be introduced via CRISPR/Cas9 mediated genome-editing. However, immaturity of iPSC-CM proves a major obstacle in their utilisation, but novel 3D culturing methods such as engineered heart tissue (EHT) have been shown to result in more mature characteristics of iPSC-CM. Titin truncation variants (TTNtv) are found in up to 25% of patients suffering from dilated cardiomyopathy, one of the major inherited cardiac conditions. There is an increased burden of ventricular arrhythmias in these patients as well as a link of TTNtv and atrial fibrillation (a form of atrial arrhythmia), indicating that TTNtv can predispose to arrhythmias in both settings.

The main scientific objective of the project is to develop iPSC-CM derived cellular model systems (both 2D and 3D) to study the predisposition to atrial and ventricular arrhythmias caused by mutations in cardiac genes. This will provide an alternative to in vivo mouse models commonly used, which can be highly invasive. TTNtv, linked to ventricular arrhythmias and atrial fibrillation in humans, will be used as example mutations. Specific aims of the project are:

1. Introduction of two pathogenic TTNtv into iPSC, done in collaboration with Professor Denning (University of Nottingham), using optimised genome-editing protocols.
2. Development of protocols to differentiate Kolf2 iPSC into ventricular and atrial iPSC-CM
   While differentiation into ventricular iPSC-CM is established in the lab, differentiation protocols for atrial iPSC-CM using retinoic acid will be established.
3. Generation of EHT from atrial and ventricular iPSC-CM. Generation of ventricular EHT is established in the lab, however atrial EHT is a novel approach to be established. Enhanced maturity of both ventricular and atrial EHT will be documented by comparison to 2D cultures of the same iPSC-CM (atrial/ventricular).
4. Characterisation of electric disturbances in iPSC-CM in the presence of TTNtv. 2D cultures of atrial and ventricular iPSC-CM with and without TTNtv will be assessed using optical mapping, multi-electrode arrays and measurements of calcium transients. Spontaneous electric activity of atrial and ventricular EHTs will be assessed by optical mapping. Challenge experiments will apply arrhythmia-inducing drugs to provoke or enhance arrhythmic behaviour.
5. Modelling of electric disturbances. Under guidance by Professor Rodriguez (University of Oxford), computational modelling will provide insight into underlying molecular changes responsible for arrhythmic behaviour. Identified targets will be validated by biochemical and electrophysiological methods.

By the end of the project the student will have a thorough understanding which arrhythmic aspects of TTNtv can be modelled in iPSC-CM derived cellular systems and how computational modelling can aide the experimental work.