Insight into cardiac fibrosis and repair through core mechanisms involving SOX9 and imbalanced ECM

Abstract

Cardiovascular diseases remain the leading causes of death worldwide despite improved interventions for diseases. The reduced mortality post myocardial infarction and pressure overload conditions has been an associated with a concurrent propagation of chronic remodelling processes which can in turn drive heart failure development. Increased understanding of the underlying pathological processes responsible for this remodelling will inform effective therapeutic development. This thesis aims to begin to uncover the role of the developmental transcription factor, SRY-Box Transcription Factor 9 (SOX9) in the stressed myocardium. The studies presented in this thesis therefore aim to identify SOX9 expression throughout cardiac development, and its role in pressure overload disease. Immunohistochemical analysis of SOX9 expression throughout human cardiac development highlights its presence in endocardial cushions and in the development of associated structures, with reduced expression upon formation of the 4-chamber heart. Immunohistochemical staining of SOX9 was also conducted following a 2-week murine model of transverse aortic constriction (TAC) in 8- to 10-week-old RosaCreER;SOX9fl/fl inducible knockout C57bl/6 mice. Echocardiography was used to record cardiac function and immunohistochemistry, qPCR and bulk-RNASeq of cardiac tissue were used to evaluate structural and molecular expression changes. It was found that 2-week TAC was sufficient to induce ectopic SOX9 expression and associated hypertrophy and fibrosis. However, in mice exhibiting SOX9 knockout these effects are no longer observed. Spatial transcriptomics (ST) technology, combining transcriptional, cellular, and spatial resolution of tissues was also employed to inform a richer understanding of the underlying pathological processes and the effects of SOX9 knockout therein. ST was performed on cryosections of TAC heart tissue and the data was primarily analysed using squidpy, a python framework, and visualised using Loupe. This allowed the identification of spatially relevant differentially expressed genes. The data was further spatially deconvoluted into cell populations and subpopulations through integration with a publicly available single-nuclear RNASeq dataset using the Cell2Location python package. This provided information regarding the distribution of disease-specific cellular subpopulations including endothelial cells, fibroblasts, and cardiomyocytes. Interestingly, the gene expression profile of the fibrosis associated cardiomyocyte subpopulations suggests a profibrotic non-canonical cardiomyocyte subtype, which is reduced when SOX9 is knocked out. Additional investigation into possible ligand-receptor interactions and the effects of the external mechanical environment begin to further uncover a potential SOX9 dependent role in altered cell states. In conclusion our study provides evidence to support a key role for SOX9 in the propagation of cardiac remodelling in both fibroblasts and cardiomyocytes. Our spatially resolved data suggests a SOX9-dependent role for profibrotic cardiomyocyte-fibroblast crosstalk in mediating cardiac disease progression.

Date of Award	31 Dec 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Elizabeth Cartwright (Supervisor), Christopher Miller (Supervisor) & Karen Piper Hanley (Supervisor)