Identification of the molecular and cellular mechanisms driving cardiac fibrosis: A novel ex vivo approach to target identification and validation

Why did we fund this project?

This joint award with the British Heart Foundation (BHF) aims to replace some rodent studies by developing a cardiac fibrosis model using human ex vivo tissue slices.

Cardiac fibrosis is a pathophysiological process present in most cardiovascular diseases. Fibrosis results from excess deposition of extracellular matrix, a mix of proteins and other molecules secreted by cells, which stiffens cardiac muscle causing a loss of cardiac cells and reduced cardiac function. There are currently no therapies available to treat cardiac fibrosis. Tissue slices are used to reduce the number of animals used in cardiac research by creating multiple slices from one heart. Experiments can then be performed on individual slices rather than individual animals. Dr Lee Borthwick and colleagues have developed a method that is able to maintain human cardiac slices in culture for up to six days replacing the use of rodent tissue slices. The student, with Lee, will now model cardiac fibrosis ex vivo by using three methods to replicate drivers of fibrosis, acute ischemia, lipid accumulation and myocardial cryoinjury, in human cardiac slices.

Cardiovascular disease (CVD) is a devastating condition attributed to ~30% of global deaths. CVD is typified by cardiac fibrosis causes reduced cardiac muscle function. Cardiac fibrosis is characterised by lipid accumulation, cardiomyocyte loss, extracellular matrix deposition and increased stiffness of the cardiac muscle. Currently no antifibrotic therapies are approved to treat CVD, highlighting an urgent need to develop new medicines. Fibrosis is a co-ordinated and dynamic process involving a wide range of cell types that drive concurrent biological processes in the complex microenvironment of human tissue. Animal models and 2D culture systems fail to recreate these complex interactions, therefore mechanisms driving fibrosis need to be interrogated in more representative, complex human tissue systems. Precision Cut Slices (PCS) from human tissue are physiologically and structurally representative of native tissue. Studying disease pathophysiology in human PCS allow us to overcome the limitations of commonly utilised model systems. My group has significantly improved PCS methodology and culture conditions to maintain human heart PCS (PCHS) for 6 days with reproducible tissue integrity and function. The extended culture allows modelling of inflammation & fibrosis and testing novel therapies in human heart tissue. In this study we will generate strong fundamental research data in clinically relevant models of human cardiac fibrosis to advance our understanding of disease pathogenesis and identify new, tractable drug targets.

To achieve this aim, we have three objectives that will have profound 3Rs impact while advancing the BHF vision:

Objective 1: Develop clinically relevant human models of cardiac fibrosis We will develop new, more clinically relevant human models of cardiac fibrosis in PCHS (acute ischemia, lipid accumulation and myocardial cryoinjury) to replace/reduce animal use and to investigate the molecular & cellular mechanisms of cardiac fibrosis.

Objective 2: Discern the molecular and cellular mechanisms of cardiac fibrosis Interrogation of novel proteomics and RNA sequencing data from PCHS will identify novel mechanisms of fibrogenesis for exploitation as therapeutic targets in CVD.

Objective 3: Testing of novel therapeutic targets in PCHS Inhibitors against novel mechanisms identified above will be explored for their ability to attenuate fibrosis in human PCHS.

Success in this project will provide validated therapeutic targets for the design of compounds that tackle the morbidity and mortality associated with CVD. Moreover, developing new human CVD tissue models to replace in-vivo models will ensure a prolonged 3Rs legacy. In the EU & UK, ~408,000 and 35,466 mice/year were used for CVD related research in 2017 and 2019. At Newcastle alone, 2,516 mice were used in cardiac injury models in 2021. If human PCHS replaced only 5% of CVD mouse procedures, this equates to a reduction of 20,400 mice/year in the EU and 1,773 mice/year in the UK. Locally, a replacement of 5% of mouse procedures would equate to a reduction of 126 mice/year. Creating PCHS from animals will further reduce animal usage. We can generate n=24 PCHS from a mouse heart and n=120 PCHS from a rat heart. Each PCHS represents an experimental repeat, drastically reducing the number of animals used per experiment. PCHS are generated from male and female mice, reducing wastage from breeding as all mice can be used for experiments. Already, integrating human lung, liver and kidney PCS into our research projects has reduced animal usage by ~60% since 2019. Our strategy to ensure continued 3Rs impact includes; presentations at national/international meetings, publication in high impact open access journals to disseminate key discoveries & share protocols, knowledge transfer via existing or new collaborations (host & train groups in the PCHS models) and collaborating with industry partners to integrate models into existing drug discovery programs.

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